JPH0824580B2 - New cosmid vector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a gene manipulation technique, and more particularly to a novel cosmid vector prepared by using the gene manipulation technique.

The gene size of mammals, including humans, often exceeds 20 kilobase pairs (hereinafter kilobase pairs are abbreviated as Kbp). Since the size of DNA that can be cloned into the widely used lambda vector is 20 Kbp, it is not possible to clone a larger gene in a functional form.

A DNA having a cohesive end site (hereinafter abbreviated as cos) of lambda phage, that is, a cosmid vector, can be used to clone a DNA having a size of 35 to 45 Kbp. Among various vectors, the cosmid vector can clone the longest DNA, but it is difficult to prepare a long genomic DNA, the background of only the vector without insert is high, and the efficiency is high. The lack of cosmid vectors has hindered the use of cosmid vectors. However, Ish-Horowicz & Burke used a phosphatase to make the vector more directional,
We succeeded in lowering the background value of the vector alone and increasing the cloning efficiency [D.Ish-Horow
icz & JFBurke, Nucleic Acids Res., 9 , 2889, (198
1)].

The method for producing a gene bank of genomic DNA by the Ish-Horowicz & Burke method is as follows (for example, currently,
PJB-, one of the widely used cosmid vectors
8 as an example). pJB-8 is a cosmid vector having a size of 5.44 Kbp, which is cleaved at one site with each of restriction enzymes BamHI, HindIII and SalI, and has one lambda cos. , PJB-8 is cleaved with HindIII to give linear DNA, and then the HindIII fragment is inactivated by phosphatase treatment. Then, two DNA fragments are obtained by cutting with BamHI, and a large DNA fragment containing cos (5.4 Kbp) is isolated by agarose gel electrophoresis. The obtained DNA fragment containing cos (5.4 Kbp) corresponds to the left arm of lambda phage. , PJB-8 is cleaved with SalI to give a linear DNA, and then treated with phosphatase to inactivate the cut piece of SalI. Then, two DNA fragments are obtained by cutting with BamHI, and a small DNA fragment containing cos (2.34 Kbp) is isolated by agarose gel electrophoresis. The obtained cos-containing DNA fragment (2.34 Kbp) corresponds to the right arm of lambda phage. , For example, the restriction enzyme Sau3A for human genomic DNA
Alternatively, cut it partially with MboI, and use D with a size of 35-45 Kbp.
Fraction NA fragments. The DNA fragment having a size of 35 to 45 Kbp is inserted between the left arm and the right arm, which are separately prepared in and, by a ligase (T-4-DNA ligase) to prepare a recombinant DNA. A gene bank of human genomic DNA can be produced by converting recombinant DNA into transduced phage particles by in vitro packaging and then infecting E. coli.

The above is the method for producing a gene bank of genomic DNA by the Ish-Horowicz & Burke method, but it has the following three problems. First, pJB-8 has only one cos and the left and right arms must be prepared separately. Secondly, DNA fragments containing cos must be separated by agarose gel electrophoresis for each of the left and right arms. Third, pJB-
The length of the left arm of 5.4 is 5.4 Kbp, the length of the right arm is 2.34 Kbp, and the lengths of the left arm and the right arm are different. You must consider the ratio.

In order to solve these problems, the present inventors have two lambda cos of the same direction,
By cutting with two kinds of restriction enzymes, we created a new cosmid vector characterized by equimolar right arm and left arm each having one cos and having almost the same length [The 36th Annual Meeting of the Japan Society for Cell Biology] Conference, Proceedings, pages 93, 94 (1983)].

The cosmid vector of the present invention has two cos having the same orientation in the same molecule, and by cutting with two kinds of restriction enzymes, each cosmid has a length of approximately one cos. Since the same right arm and left arm can be equimolar, it is not necessary to separately prepare the left arm and the right arm, and it is not necessary to separate them by agarose gel electrophoresis. Furthermore, since the lengths of the right arm and the left arm are almost the same, it can be considered that the degree of ligation to the genomic DNA of 35 to 45 Kbp by T4-DNA ligase is almost the same, and it was cut with two kinds of restriction enzymes. If the equimolar mixture of the right arm and the left arm is ligated with 35-45 Kbp of genomic DNA as it is, it can be expected that half of the recombinant DNA can be transformed into transduction phage particles. Thus, when the vector of the present invention is used, a DNA fragment of 35 to 45 Kbp can be obtained as a genomic DN.
Gene bank A can be manufactured more easily and efficiently.

PDcos Ap, which is one of the novel cosmid vectors of the present invention
^r / ori is shown in Fig. 1 [36th Annual Meeting of the Japanese Society for Cell Biology,
Abstracts 93 pages (1983)]. pDcos Ap ^r / ori is unidirectional from the charge vector Charon4A
2 cos and neomycin resistance gene (ne from Tn903)
o ^r ) 〔Oka, Sugisaki & Takanami, J. Mol. Biol., 147 , 217
(1981)], an ampicillin resistance gene (Ap ^r ) derived from pBR-327, and an origin of replication (ori) [Soberon, Cavarrubias.
& Bolivar, Gene, 9 , 287 (1980)]. The size is 4.7 Kbp, 1 site for each of the restriction enzymes PvuII, Bam HI and PstI, 2 sites for the restriction enzyme Bst EII,
It is cleaved at 3 sites by the restriction enzyme Eco RI. This vector has the following features. , There are two cos with the same direction. , Bam HI site with Sau3A or
It is possible to clone 35-45 Kbp genomic DNA that has been digested with MboI or the like. The insert can be excised from the vector by cutting the Eco RI sites on both sides of the Bam HI site. , PvuII cleaves the vector and treats it with phosphatase to give the vector directionality, and by cleaving the Bam HI site, equimolar right and left arms are created. The T-DNA ligase was used to ligate the right and left arms of the vector with Bam HI sites on both sides of the 35-45 Kbp genomic DNA that had been digested with Sau3A or MboI, and the recombinant DNA was subjected to in vitro packaging. It can then be transformed into transduced phage particles and then infected with E. coli. This transductant DNA
Contains the vector-derived Ap ^r and ori, so that by selecting with ampicillin, only E. coli having the recombinant DNA as a plasmid can be selectively utilized.
The method for producing a gene bank of genomic DNA (foreign DNA) using this vector is as follows (see FIG. 2). , PDcos Ap ^r / ori is digested with PvuII to form linear DNA, and then treated with phosphatase to produce PvuII.
Inactivate the II cut piece. Then, by cutting with Bam HI, a right arm and a left arm each having one cos and having substantially equal lengths are prepared. Then, the foreign DNA is limitedly digested with Sau3A or MboI to prepare a DNA fragment with a size of 35 to 45 Kbp. A recombinant DNA is prepared by inserting a 35-45 Kbp genomic DNA fragment between the left arm and the right arm prepared by using T4-DNA ligase. , Recombinant DNA in
In vitro packaging changes to transduced phagy particles,
Infect E. coli. By selecting the transduced ampicillin-resistant Escherichia coli with ampicillin, only Escherichia coli having the recombinant DNA as a plasmid can be utilized. In this way, a gene bank of genomic DNA can be manufactured.

In addition, the present inventors have succeeded in creating a cosmid vector, pDcos Tc ^r (Fig. 3) and pDcos Tc ^r / ori (Fig. 4) for recovering genes from transformant cells (36th Japan). Cell biology society conference, Proceedings, p. 94 (1983)]. Mammalian marker genes can be isolated by using gene transfer into cultured mammalian cells. There is an operation for recovering the prepared DNA sequence from the transformant cells into Escherichia coli, but the novel cosmid vector pDcos Tc included in the present invention is included.
^r and pDcos Tc ^r / ori are useful vectors for this gene recovery. pDcos Tc ^r consists of 2 cos having the same directionality derived from Charon4A, a tetracycline resistance gene (Tc ^r ) derived from pBR-327, and an origin of replication (ori).
pDcos Tc ^r / ori, as well as the cos2 pieces, pBR-327-derived Tc ^r
And ori and SV-dihydrofolate reductase gene (SV-dhfr) [Subramani, Mulligan &
Berg, Mol. Cell. Biol., 1,854 (1981)]. These vectors are based on pDcosAp ^r / ori described above (Fig. 1).
It has the same characteristics as those of Bam H and can be used to produce a gene bank of genomic DNA.
The only difference is that instead of the I site, a 35 to 45 Kbp genomic DNA that has been digested with Sau3A or MboI can be cloned into the BglII site. Since the transduction phage particles created in pDcos Tc ^r does not have the ori of the plasmid, it can not grow even if infected with E. coli. Ori near the gene of interest
The target gene can be recovered in Escherichia coli as a tetracycline-resistant (Tc ^r ) clone only in the presence of such a gene. On the other hand, the transduction phage particles prepared with pDcosTc ^r / ori have ori and Tc ^r markers, and a gene bank of genomic DNA can be prepared as a Tc ^r clone. If there is a drug resistance marker such as Ap ^{r in} the vicinity of the gene of interest, only the desired one can be selectively recovered in E. coli as a drug resistance clone such as ampicillin resistance (Ap ^r ).

Furthermore, we have have a selection marker for mammalian cells, cosmid vectors for the purpose of gene transfer into mammalian ^{cells, pDcosAp r / ori / gpt /} TK ( Fifth
Figure) was created. pDcosAp ^r / ori / gpt / TK is cos2 with the same direction derived from Charon4A, Ap ^r and ori derived from pBR-327, and SV-dhfr derived from pSV2-dhfr as a spacer.
Gene, pSV2-gpt-derived gpt gene [Richard, Mulligan
& Berg, Mol. Cell. Biol., 1, 449 (1981)], and pTK-
4 derived TK gene [Ishiura, M., et al., Mol. Cell. Biol.,
2 , 607 (1982)]. This cosmid vector, like pDcosAp ^r / ori described above, can produce the gene bank of genomic DNA. That is, there are two cos with the same direction, and by cutting with PvuII and BamHI, the right arm and the left arm having one cos and having almost the same length can be equimolar, and the BamHI site has 35 cos. ~ 45 Kbp genomic DNA
It is possible to create a recombinant DNA in which is inserted. Then, this recombinant DNA can be transformed into transduced phage particles by in vitro packaging and transferred to Escherichia coli. Since the recombinant DNA contains Ap ^r and ori derived from the vector, it contains the recombinant DNA. E. coli can be obtained as an ampicillin resistant clone by selecting with ampicillin. Furthermore, since this recombinant plasmid has the gpt gene and the TK gene, the xanthine guanine phosphoribosyl tranferase (gpt) activity or the thymidine kinase (TK) activity can be used as a marker when the gene is transferred into cultured mammalian cells. Therefore, there is an advantage that the insertion of the recombinant DNA derived from the vector can be determined at the level of the cultured cells.

Examples of cos used in the present invention include those derived from lambda or lambda. In particular, cos derived from lambda phage is mentioned as a preferable one.

The vector of the present invention has a right arm and a left arm each having one cos by cutting with two kinds of restriction enzymes. As a combination of these two kinds of restriction enzymes, PvuII and BamHI, PvuII and BglII, ClaI and BamHI and ClaI
BglII etc. can be mentioned. Of course, other combinations can be considered, and the invention is not limited to these.

In order to determine that the vector-derived DNA fragment has been inserted into the vector of the present invention, selection by drug resistance is generally performed. Therefore, it is preferable that the vector of the present invention also incorporates a DNA fragment expressing drug resistance. Examples of drug resistance include ordinary antibiotic resistance such as ampicillin resistance, tetracycline resistance, neomycin (kanamycin) resistance, or chloramphenicol resistance. pDcos Ap ^r / ori has ampicillin resistance gene and neomycin resistance gene, pDcosT
c ^r and pDcosTc ^r / ori has a tetracycline resistance gene. pDcosAp ^r / ori / gpt / TK is with the ampicillin resistance gene.

When a gene bank of genomic DNA (foreign DNA) is produced using the vector of the present invention and the gene (gene bank) is transferred to cultured mammalian cells, it is determined that the vector-derived DNA fragment has been inserted. Therefore, it is preferable that a DNA fragment expressing a specific physiological function is incorporated in the vector of the present invention. Aminoglycoside 3'-phosphotransferase (neo) gene [Oka, Sugisaki & Takanami, J. Mol.
Biol., 147, 217 (1981 ) ], thymidine kinase (TK) gene [Wigler, M.etal., Cell, 14 , 725 (1978), adenine phosphoribosyl trans Hue hydrolase (APRT) gene [Wi
gler, M., etal., Pro.Natl.Acad.Sci.USA, 76 , 1373 (19
79)], hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene [Graf, LHJr., Et al, Som
at.Cell.Genet., 5 , 1031 (1979)], xanthinganine phosphoribosyl transferase (XGPRT or gpt)
Gene [Berg, P., et al. Mol. Cell. Biol., 1,449 (198
1)] and the like. New cosmid vectors pDcosAp ^r / ori / gpt / TK of the present invention has a gpt gene and the TK gene.

Further, the vector of the present invention preferably has a gene fraction having a replication function derived from ColEI.

Examples of the foreign DNA used in the present invention include DNA derived from microorganisms such as bacteria, filamentous fungi, yeasts, higher animals and plants, various types of phages, and the like.

In addition, there is a complementary relationship between the restriction enzyme used in the present invention (enzyme having a function of selectively cutting a specific base sequence site of a DNA fragment) or a DNA sticky end formed by cutting with a restriction enzyme. An enzyme (ligase) used to bond the cohesive ends of other DNA fragments, or an enzyme that makes the cohesive ends of DNA ends complementary to each other to bond DNA ends that are not complementary to each other, or a ligase that connects these Is already well known, many enzymes are commercially available, and methods using them are widely disclosed [Molecular Cloning, Cold
Spring Harbor Laboratory, Maniatis, T., etal., (198
2)], so you can use them.

By using the vector of the present invention, it will be easier to analyze cancer and genetic diseases of mammals including humans at the molecular level. For example, a gene bank of genomic DNA derived from normal human cells is used as pDcos Ap ^r
/ ori, and transform (normalize) genetically diseased cells with this gene bank to obtain transformant cells. Genomic DNA was extracted from transformant cells and p
Create a gene bank of genomic DNA with DcosTc ^r or pDcosTc ^r / ori and recover the target gene. By examining the target gene from various aspects, human genetic diseases can be analyzed at the molecular level.

In addition, using the vector of the present invention, genomic DNA (foreign DN
By creating the gene bank of A), it is possible to efficiently find a specific gene that expresses a useful substance. This specific gene includes DNA derived from bacteria, filamentous fungi, microorganisms such as yeast, higher animals and plants or various types of phages, and specific useful substances include, for example, various hormones, enzymes, and further antibiotics and amino acids. And other synthetic enzymes.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 PDcosTc ^r creation and preparation (Figure 6) (a) pAPcos creation and cos derived Charon4A is prepared i) creating full Urge vector is a cosmid vector having one pHC79 [Hohn & Collins, G
ene, 11 , 291 (1980)] 20 μg to the reaction mixture 200 μl (10 m
MTris-HCl (pH7.6), 12mM MgCl 2, 10m MDTT, 100μg /
Completely digest with 29.9 units of restriction enzyme BalI (New England Biolabs) at 37 ° C for 2 hours with ml BSA (bovine serum albumin), and then add 500 µ of the reaction mixture.
1 (150 mM NaCl, 6 mM Tris-HCl (pH 7.9), 6 mM MgCl ₂ ,
6mMDTT, 100μg / μl BSA], 41.1 unit restriction enzyme
Bst EII (manufactured by New England Biolabs) was reacted at 60 ° C. for 2 hours for complete digestion. After the reaction was completed, extraction was performed with an equal volume of water-saturated phenol, the aqueous layer was concentrated with secondary butanol, 0.1 volume of 3M sodium acetate and 2 volume of ethanol were added to obtain DNA as a precipitate, and 95% ethanol was added. After washing with water, it is dried with a water pump to prevent precipitation.
It was dissolved in 14 μl (up to 1 μg / μl) of an aqueous solution (hereinafter abbreviated as 10 t / 0.1E) consisting of 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA.

Next, 1 μl of this DNA solution digested with BalI and BstEII
(~ 1 μg) was added to the reaction mixture 25 μl [50 mM Tris-HCl (pH 7.
2), 10 mM MgSO ₄ , 0.1 mM DTT, 50 μg / ml BSA, 80 μM dCT
R, 80 μM dGDP, 80 μM dATP, 80 μM dTTp] and 2 units of Klenow enzyme (Boehringer Mannheim) at 22 ° C.
After reacting for 0 minutes, a double-stranded DNA fragment (b) having complementary ends to each other has a double-stranded DNA fragment (cohesive end) aligned (b).
lunt end). After completion of the reaction, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme.

Then, add 10 μl of this solution (DNA ~ 0.4 μg) to the reaction mixture 50
μl (50 mM Tris-HCl (pH7.4), 10 mM MgCl ₂ , 10 mM DT
T, 1 mM spermidine, 1 mM ATP, 100 μg / ml BSA), 15
T4-DNA ligase (reacted manufactured by New England Biolabs and 17.5 hours at 12 ° C. of Yunitsuto was cos and ori, DNA fragments containing Ap ^r ₁ Tc ^r a (4.75Kbp) is closed reduction. After the completion of the reaction, phenol After extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

ii) Transformation 10 μl of this DNA solution was added to competent cell 1 of Escherichia coli HB101.
00μl (logarithmic growth E. coli HB101 at 0-4 ° C with 0.1M Ca
After treatment with Cl ₂ solution for 15 minutes, 0.1 / 10 of the original culture solution was added.
Resuspend in M CaCl ₂ /14% glycerol solution and store at -70 ° C)
After mixing well with 0 ° C for 10 minutes and then at 37 ° C for 5 minutes to take up the DNA into E. coli, L-
Broth (1% Bactotryptone, 0.5% Yeast Extract 0.5% NaC
2 ml of 0.1% glucose) was added, and the mixture was cultured at 37 ° C. for 1 hour with shaking. This cell suspension was treated with 20 μg / ml ampicillin and
L-plate containing 20 μg / ml tetracycline (L-
Sprinkle the broth on a plate containing 1% agar, 37
After culturing at 48 ° C for 24-48 hours, ampicillin- and tetracycline-resistant clones were obtained.

iii) Small amount culture and small amount DNA preparation Ap ^r ₁ Tc ^r clone was added to 2 ml of L-broth (Ap 20 μg /
The cells were grown by shaking at 37 ° C. (containing ml) overnight. Culture medium 1.
Take 5 ml of Eppendorf tube and collect the cells in a desktop centrifuge, and extract the cells (8% sucrose, 0.5%).
% TritonX-100, 50 mM EDTA (pH8.0), 10 mM Tris-HCl
(PH 8.0)) and resuspended in 0.35 ml. Next, 25 μl of a lysozyme solution [10 mg / ml, 10 mM Tris-HCl (pH 8.0)] was added and mixed, and then the tube was placed in boiling water for 40 seconds.
Then immediately put the tube in a centrifuge (21,000
The supernatant was obtained at 50 rpm for 15 minutes, and 0.5 μl of RNase A solution (20 mg / ml, 10 t / 0.1E) was added to the supernatant, and the mixture was kept at 37 ° C. for 1 hour to decompose RNA. Then, after deproteinizing with phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was extracted with 10 t / 0.1
It was dissolved in 20 μl of E to obtain a plasmid DNA solution.

iv) Characterization of plasmid DNA Then, 1 μl or 2 μl of this plasmid DNA solution was digested with restriction enzymes EcoRI, BstEII, BglII, PvuII, etc., and then 0.4-1.5% agarose gel electrophoresis [40 mM Tris, 20
mM sodium acetate, 2 mM EDTA (pH8.15); 10 mA, 12-24 hours]. Degraded DNA fragments can be detected by ethidium bromide staining and long wavelength UV illumination. In this way, the plasmid DNA carried by each clone was characterized by constructing a restriction map of the plasmid DNA. And D between the BalI site and the BstEII site of PHC79
Plasmid lacking NA fragment (1.68 Kbp): pAPcos (6th
E. coli having (Fig.) Was obtained.

V) Mass culture and preparation of plasmid DNA This E. coli was cultured in TYG-P medium (1% Bactotryptone, 0.1%).
% Yeast extract, 0.2% glucose, 0.1% NH ₄ Cl, 0.3%
NaCl, 0.01% Na ₂ SO ₄ , 0.08% MgCl ₂ · 6H ₂ O, 1.52% Na ₂
HPO ₄・ 12H ₂ O, 0.3% KH ₂ PO ₄ ) 1 (containing Ap 20 μg / ml), shake culture at 37 ℃, the absorbance at 600 nm (A600)
Chloramphenicol at 100 μg / ml at 1.0
Was added so that the concentration would become, and the mixture was further cultured by shaking at 37 ° C. for 15 hours. After culturing, centrifuge (6,000 rpm, 10 minutes)
The cells were harvested over time, and then the cells were washed with 25% sucrose and 50 mM T.
It was resuspended in 18 ml of isotonic buffer consisting of ris-HCl (pH 8.0). Add lysozyme solution (5 mg / ml, 0.25M Tris-HC
l (pH8.0)] 3.6 ml, gently mix and stir at 0 ° C for 5 minutes, then add 0.25M EDTA (pH8.0) solution 7.2 ml, gently mix and stir at 0 ° C for 5 minutes I was there. afterwards,
2.7 ml of 10% SDS solution and 8.1 ml of 5M NaCl solution were added and gently mixed, and the mixture was allowed to stand at 0 ° C. overnight and then centrifuged (20,000
rpm, 45 minutes) to a crude lysate ~ 39
I got ml. RNase A solution (20 mg / ml, 10 t /
After adding 20 μl of 0.1E) and incubating at 37 ° C. for 1 hour to decompose RNA, deproteinization was carried out by phenol extraction, and the gel was filtered with Sepharose 2B. Plasmid DNA
DNA was obtained as an ethanol precipitate from a void fraction containing EDTA, and then the DNA was cesium chloride-ethidium bromide solution (1M Tris-HCl (pH 8.0) 0.375 ml, 0.25 M EDTA (pH 8.
_{0) 0.30ml, H 2 O6.45ml,} CsCl7.5g, EtBr solution (4 mg / ml)
0.375 ml] and subjected to equilibrium density gradient centrifugation (60,000 rpm, 12 hours). Then, the covalently closed circular plasmid DNA (ccc-DNA) appears as a band under the fluorescent band of the linear plasmid DNA and the chromosome in the centrifuge tube under long-wavelength ultraviolet irradiation. . This ccc-DNA band was fractionated and extracted with isopropanol saturated with a CsCl aqueous solution to remove endium bromide, then 10 t / 0.
Dialysis was performed on 1E 1. Then, ccc-DNA was obtained as an ethanol precipitate from the dialysate, and the precipitate was 10 t / 0.
Dissolve in 2 ml of 1E and pAPcos plasmid DNA solution (0.418 m
g / ml) was prepared.

(B) Preparation and preparation of pAPcosΔ (EcoRI) 10 μg of pAPcos was mixed with 100 μl of the reaction mixture (100 mM Tris-HCl).
(PH 7.5), 50 mM NaCl, 5 mM MgCl ₂ , 100 μg / ml BSA)
The 60.6 unit restriction enzyme EcoRI (New England Biola
bs) and reacted at 37 ° C. for 2 hours for complete digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 7 µl (~ 1 µg / µl).

Next, 1 μl of this DNA solution digested with EcoRI (up to 1 μl
g) to the reaction mixture 25 μl (50 mM Tris-HCl (pH7.2), 10 m
M MgSO ₄ , 0.1 mM DTT, 50 μg / ml BSA, 80 μM dATP, 80 μM
dCTP, 80 μM dGTP, 80 μM dTTP) with 2 units of Klenow
React with enzyme for 30 minutes at 22 ℃ and blunt the cohesive end.
I changed it to end. Then, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme.

Next, add 10 μl (~ 0.4 μg) of this DNA solution to the reaction mixture 50
μl (50 mM Tris-HCl (pH7.4), 10 mM MgCl ₂ , 10 mM DT
T, 1 mM spermidine, 1 mM ATP, 100 μg / ml BSA) was reacted with 15 units of T4-DNA ligase at 12 ° C. for 17.5 hours for cyclization, followed by phenol extraction to obtain DNA as an ethanol precipitate. The precipitate was 0.1 MT ris. -HCl (pH 7.5) solution 2
It was dissolved in 0 μl.

After transformation with 10 μl of this DNA solution with E. coli (HB101), Ap ^r and Tc ^r clones were obtained. Next, small amount culture and small amount of these clones
After DNA preparation, plasmid D of each clone
By examining the characteristics of NA, Escherichia coli harboring a plasmid lacking the EcoRI site of pAPcos: pAPcosΔ (EcoRI) (Fig. 6) was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pAPcosΔ (EcoR
The plasmid DNA of I) was prepared.

(C) Preparation and preparation of pHPcos 20 μg of pAPcosΔ (EcoRI) was added to 200 μl of the reaction mixture [60 mM.
NaCl, 6mM Tris HCl (pH8.0) , 10mM MgCl 2 6mM DTT, 10
0 μg / ml BSA] with 75.8 unit restriction enzyme AvaI (New Engl
and Biolabs) at 37 ° C for 2 hours for complete digestion, and then 500 µl of the reaction mixture [60 mM NaCl, 7 mM T
ris-HCl (pH7.4), 7 mM MgCl ₂ , 100 μg / ml BSA],
The product was completely digested by reacting with the unit restriction enzyme HindIII (New England Biolabs) at 37 ° C. for 2 hours. Then, after deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 14 μl (˜1 μg / μl).

Next, 1 μl of this DNA solution digested with AvaI and HindIII
(~ 1 μg) in 25 μl of reaction mixture, 2 units of Klen
ow the enzyme with ow enzyme for 30 minutes at 22 ℃ and blun the cohesive end.
changed to t end. Then, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme.

Next, add 10 μl (~ 0.4 μg) of this DNA solution to the reaction mixture 50
After reacting with 15 μl of T4-DNA ligase at 12 ° C. for 17.5 hours, the DNA fragment containing cos, ori and Ap ^r (3.75 Kb
p) was cyclized (HindIII site was reconstructed).
Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 20 μm.
It was dissolved in 1.

After transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^s (tetracycline sensitive) clones were obtained. Next, the characteristics of the plasmid DNA possessed by these clones were examined, and pAPcosΔ (EcoR
I) between the AvaI site and the HindIII site (1 Kb
Escherichia coli having a plasmid lacking p): pHPcos (Fig. 6) was obtained.

After growing this E. coli in TYG-P medium 1,
Ccc-DNA was isolated and purified from the bacterial cells to obtain the plasmid DN of pHPcos.
A was prepared.

(D) Preparation and preparation of pHBglIIcos 10 μg of pHPcos was mixed with 100 μl of reaction mixture [60 mM NaCl, 6 mM T
ris-HCl (pH 7.5), 6 mM MgCl ₂ 6 mM DTT, 100 μg / ml BS
A] with 51.2 unit restriction enzyme PvuII (Takara)
The reaction was carried out at 37 ° C for 2 hours for complete digestion. (Blunt en
d). After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1E 7 µl (~ 1 µg / µ
l).

Next, 0.4 μl of the DNA solution digested with PvuII (up to 0.4 μl
g) and a BglII linker [d (pC-A-G-A-T-C-
TG)] 2 μg and 20 μl of reaction mixture [50 mM Tris-H
Cl (pH7.4), 10 mM MgCl ₂ , 10 mM DTT, 1 mM spermidine,
1 mM ATP, 100 μg / ml BSA] and ligated by reacting with 6 units of T4-DNA ligase at 22 ° C. for 6 hours. After that, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was added to 250 μl of the reaction mixture [60 mM NaCl, 10 mM T
ris-HCl (pH7.4), 10 mM MgCl ₂ , 10 mM DTT, 100 μg / ml BS
A], 200 units of restriction enzyme BglII (Takara) and 3
After reacting at 7 ° C for 4 hours for digestion, the mixture was applied to 0.8% preparative agarose gel and electrophoresed at 10 mA for 12 hours. Degraded DNA fragments are visible by ethidium bromide staining and long-wavelength UV illumination. A gel containing a DNA fragment (2.55 Kbp) consisting of cos, ori, and Ap ^r was cut out, and then extracted with 5 volumes of an extract (0.5 M NH ₄ OAc, 1 mM EDTA (pH 7.5): water-saturated phenol = 1 :). In the presence of 1), mash the gel and D
NA was eluted. Then, the eluate was concentrated with secondary butanol, and 0.1 times volume of 3M sodium acetate and 2 times volume of ethanol were added to obtain DNA as a precipitate.

This DNA precipitate was treated with 20 μl of the reaction mixture to prepare 6 units of T4-D.
After reacting with NA ligase at 12 ℃ for 17.5 hours, cos and ori, A
The DNA fragment consisting of p ^r (2.55 Kbp) was closed. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Next, the characteristics of the plasmid DNA of these clones were examined to obtain Escherichia coli having the plasmid: pHBglIIcos (Fig. 6) in which the BglII linker was inserted into the PvuII site of pHPcos.

After growing this E. coli in TYG-P medium 1,
The ccc-DNA was separated and purified from the cells to prepare a pHBglIIcos plasmid DNA.

(E) Preparation and preparation of pAPcos (BglII) 10 μg of pAPcos was added to 100 μl of the reaction mixture to obtain 60.6 units of E.
It was digested by reacting with coRI at 37 ° C for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 7 µl (~ 1 µg / µl).

Next, 1 μl of this DNA solution digested with EcoRI (up to 1 μl
g) in a reaction mixture of 25 μl with 2 units of Klenow enzyme
The reaction was performed at 22 ° C for 30 minutes, and the cohesive end was changed to blunt end. Then heat the reaction mixture at 65 ° C for 10 minutes and heat it to Klenow.
The enzyme was inactivated.

Then, 10 μl (0.4 μg) of this DNA solution and 2 μg of BglII linker [d (pC-A-G-A-T-C-T-T-G)] were mixed with 20 μl of a reaction mixture to prepare 6 units of T4-DNA ligase. Was reacted at 22 ° C. for 6 hours for ligation.

Then, add 10 μl of this reaction solution to 50 μl of the reaction mixture,
After reacting with unit T4-DNA ligase at 12 ° C. for 17.5 hours, cos and ori, Ap ^r . DNA fragment consisting of Tc ^r (4.75Kbp)
Was closed. After that, phenol extraction was performed
Was obtained as an ethanol precipitate, and the precipitate was mixed with 0.1 M Tris-HCl (pH
7.5) Dissolved in 20 μl of solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^r clones were obtained. Then, the characteristics of the plasmid DNA of these clones were examined to obtain Escherichia coli having the plasmid: pAPcos (BglII) (Fig. 6) in which the BglII linker was inserted into the EcoRI site of pAPcos.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pAPcos (BglII).
Was prepared.

(F) Preparation and preparation of pDcos Ap ^r / Tc ^r (2BglII) 20 μg of pAPcos (BglII) was reacted with 202 units of BglII in 200 μl of the reaction mixture at 37 ° C. for 2 hours for digestion, and then 0.8% for preparation agarose gel. Electrophoresis, cos
And it was separated and purified DNA fragment (1.86Kbp) containing Tc ^r, DNA
Is obtained as an ethanol precipitate, and the precipitate is 10t / 0.1E 5.5 μl
(˜1 μg / μl).

On the other hand, 10 μg of pHBglIIcos was digested by reacting 100 μl of the reaction mixture with 94 units of BglII at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 7 µl (∼1 µg / µl).

Then, BglII and PAPcos DNA solution 1μl of BglII fragment containing the cos and Tc ^r of (BglII) (~1μg), the pHBglIIcos
1 μl (~ 1 μg) of the DNA solution digested with
It was ligated by reacting with 0 μl of 6-unit T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was carried out to obtain DNA as an ethanol precipitate, and the precipitate was extracted with 0.1 M Tris-
It was dissolved in 20 μl of HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^r clones were obtained. Then, the properties of the plasmid DNAs of these clones were examined to obtain Escherichia coli having a plasmid having two cos having the same orientation: pDcos Ap ^r / Tc ^r (2BglII) (Fig. 6).

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / Tc.
^r (2BglII) plasmid DNA was prepared.

^{(G) pDcos Ap r / Tc} r creation and preparation ^{pDcos Ap r / Tc r (2BglII} ) 10μg reaction mixture 100μl
Then, it was reacted with 11.0 unit of BglII at 37 ° C for 30 minutes for limited digestion. Next, after deproteinizing with phenol extraction, DNA was obtained as an ethanol precipitate. This DNA precipitate is added to the reaction mixture 100
After reacting with 2 μl of 21.8 unit PvuII for 2 hours at 37 ° C for complete digestion, it was subjected to 0.8% preparative agarose gel electrophoresis and subjected to a DNA fragment containing two cos and ori, Ap ^r , and Tc ^r (4.2 Kbp). 2) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 6.5 t (0.1 μg / μl) of 10 t / 0.1E.

Next, 1 μl (~ 1 μg) of this DNA solution was added to the reaction mixture 2
5 μl was reacted with 2 units of Klenow enzyme at 22 ° C. for 30 minutes to change cohesive end to blunt end. And
The reaction solution was heat-treated at 65 ° C for 10 minutes to inactivate the Klenow enzyme.

Next, 10 μl (0.4 μg) of this DNA solution and 2 μg of PvuII linker [d (pC-C-A-G-C-T-TG-G)] were mixed with 20 μl of a reaction mixture to prepare 6 units of T4-DNA ligase. Was reacted at 22 ° C. for 6 hours for ligation.

Then, add 10 μl of this reaction solution to 50 μl of the reaction mixture,
After reacting with unit T4-DNA ligase at 12 ℃ for 17.5 hours, a DNA fragment consisting of two cos and ori, Ap ^r , and Tc ^r (4.2K
bp) was closed. Then, phenol extraction was carried out to obtain DNA as an ethanol precipitate, and the precipitate was extracted with 0.1 M Tris-
It was dissolved in 20 μl of HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^r clones were obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and a plasmid lacking the DNA fragment (0.2 Kbp) between the PvuII site and the BglII site of pDcos Ap ^r / Tc ^r (2BglII): pDcos Ap ^r / Tc ^r E. coli having (Fig. 6) was obtained.

This E. coli was grown in TYG-P medium 1, and ccc-DNA was separated and purified from the cells to prepare pDcos Ap ^r / Tc ^r plasmid DNA.

(H) in ^{pDcos Ap r / Tc r · XhoI} a creation and preparation pDcos Ap ^r / Tc ^r 10μg reaction mixture 100 [mu] l, was digested by reacting for 2 hours at 42.9 Yunitsuto of AvaI and 37 ° C.. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 7 µl (~ 1 µg / µl).

Next, 1 μl of DNA solution digested with this AvaI (up to 1 μl
g) in a reaction mixture of 25 μl with 2 units of Klenow enzyme
The reaction was performed at 22 ° C for 30 minutes, and the cohesive end was changed to blunt end. Then heat the reaction mixture at 65 ° C for 10 minutes and heat it to Klenow.
The enzyme was inactivated.

10 μl (0.4 μg) of this DNA solution and XhoI linker [d
(PC-C-T-C-G-A-G-G)] 2 μg was reacted with 6 units of T4-DNA ligase in 20 μl of the reaction mixture at 22 ° C. for 6 hours for ligation. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was mixed with 600 μl of the reaction mixture (150 mM NaCl, 6 mM T
ris-HCl (pH 7.9), 6 mM MgCl ₂ , 6 mM DTT, 100 μg / ml BS
In A), 1000 units of XhoI (manufactured by New England Biolabs) were reacted at 37 ° C. for 4 hours for digestion and then subjected to 0.8% preparative agarose gel electrophoresis to give two cos and o.
A DNA fragment (4.2 Kbp) containing ri, Ap ^r and Tc ^r is separated and purified,
DNA was obtained as an ethanol precipitate.

This DNA precipitate was treated with 20 μl of the reaction mixture to prepare 6 units of T4-D.
After reaction with NA ligase at 12 ° C. for 17.5 hours for cyclization, folloWed by phenol extraction, DNA Was obtained as an ethanol precipitate, and the precipitate Was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^r clones were obtained. In the next, by examining the characteristics of plasmid DNA possessed by these clones, the plasmid was inserted the XhoI linker AvaI site pDcos Ap ^r / Tc ^r: with ^{pDcos Ap r / Tc r · XhoI} a ( Figure 6) E. coli was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / Tcr ^r.
XhoI ^a plasmid DNA was prepared.

(I) pDcos Tc ^r creation and preparation ^{pDcos Ap r / Tc r · XhoI} a 10μg in the reaction mixture 100 [mu] l 6.
Two units of BstEII were reacted at 60 ° C for 30 minutes for limited digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1 E7 μl (~ 1 μg / μ
l).

Next, 1 μl of the DNA solution that had been digested with this BstEII (
1 μg) with 25 μl of the reaction mixture and reacted with 2 units of Klenow enzyme at 22 ° C for 30 minutes to blunt en the cohesive end.
changed to d. Then, heat the reaction solution at 65 ° C for 10 minutes to remove Kle.
now the enzyme has been deactivated.

10 μl (0.4 μg) of this DNA solution and PstI linker [d
(PG-C-T-G-C--A-G-C)] 2 μg,
20 μl of reaction mixture and 6 units of T4-DNA ligase and 22 ℃
Was reacted for 6 hours and ligated. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was added to 400 μl of the reaction mixture (50 mM (NH ₄ ) ₂ SO ₄ , 2
0 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 100 μg / ml BSA)
And a 600 unit PstI (New England Biolabs)
After 4 hours reacted digested with 37 ° C., agarose gel electrophoresis 0.8% preparation, two cos and ori, Tc ^r
Was isolated and purified to obtain DNA as an ethanol precipitate.

Next, this DNA precipitate was reacted with 6 μl of T4-DNA ligase at 12 ° C. for 17.5 hours for cyclization in 20 μl of the reaction mixture, followed by phenol extraction to obtain DNA as an ethanol precipitate. -HCl (pH 7.5) solution 20
It was dissolved in μl.

Then, after transforming Escherichia coli (HB101) with 10 μl of this DNA solution, a Tc ^r clone was obtained. Then, the characteristics of the plasmid DNA of these clones were examined, and the plasmid pDcos Ap ^r / Tcr ^r XhoI ^a lacking the DNA fragment (~ 0.2 Kbp) between the BstEII and PstI sites: pD
E. coli with cos Tc ^r (Figs. 3 and 6) were obtained. In addition, this strain was submitted to the Microtechnology Research Institute
No. 51 (FERMP-7551) "has been deposited.

The E. coli, after grown in TYG-P medium 1, and plasmid DNA was prepared for pDcos Tc ^r a ccc-DNA from the cells was separated and purified.

Example In 2 pDcos Tc ^r / ori creation and preparation (Figure 7) (a) pBR-327 · XhoI p creation and preparation pBR-327 10 [mu] g of reaction mixture 100 [mu] l, 24.8 Yunitsuto of PstI and 37 2 hours at ℃ It was reacted and digested. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 7 µl (~ 1 µg / µl). Next, 1.5 μl of this PstI-digested DNA solution (~ 1.5
30 μl of reaction mixture (33 mM Tris-aceta-te (pH
7.9), 66 mM potassium acetate, 10 mM MgCl ₂ , 0.5 mM DTT, 100 μg /
me BSA, 0.1mM dATP, 0.1mM dCTP, 0.1mM dGTP, 0.1mM
dTTP), react with 5 units of T4-DNA polymerase (manufactured by PL) at 37 ° C for 15 minutes to blun the cohesive end.
changed to t end. Then, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate T4-DNA polymerase.

8 μl (0.4 μg) of this DNA solution and XhoI linker [d
(PC-C-T-C-G-A-G-G)] 2 μg was reacted with 6 units of T4-DNA ligase in 20 μl of the reaction mixture at 22 ° C. for 6 hours for ligation. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was treated with 600 μl of the reaction mixture to obtain 1000 units of Xh.
After digestion by reacting with oI for 4 hours at 37 ° C., it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify the 3.27 Kbp DNA fragment, and the DNA was obtained by ethanol precipitation.

This DNA precipitate was mixed with 20 µl of the reaction mixture to prepare 6 units of T4-DNA.
After cyclization by reacting with ligase at 12 ° C for 17.5 hours,
A DNA was obtained as an ethanol precipitate by phenol extraction, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

Then, after transforming Escherichia coli (HB101) with 10 μl of this DNA solution, a Tc ^r clone was obtained. Then, the characteristics of the plasmid DNA of these clones were examined to obtain Escherichia coli harboring the plasmid: pBR-327.XhoI ^p (Fig. 7) in which the XhoI linker was inserted into the PstI site of pBR-327.

This E. coli was grown in TYG-P medium 1, and ccc-DNA was separated and purified from the cells to obtain pBR-327.XhoI ^p.
Was prepared.

(B) Preparation and preparation of pDcos Tc ^r (2ori) 20 μg of pDcos Tc ^r was added to 500 μl of the reaction mixture (150 mM NaCl,
6mM Tris-HCl (pH7.9), 6mM MgCl 2, 6mM DTT, 100μg / m
l BSA), 167 unit restriction enzyme BamHI (New England
Biolabs) and 500 units of XhoI at 37 ° C for 2 hours for complete digestion, and then 0.8% preparative agarose gel electrophoresis for DNA containing two cos and ori.
The fragment (2.4 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 9.5 μl of 10 t / 0.1E (˜1 μg / μl).

On the other hand, 20 μg of pBR-327 XhoI ^{p in} 500 μl of the reaction mixture,
167 units of BamHI and 500 units of XhoI at 37 ℃
After reacting for a period of time to complete digestion, 0.8% preparative agarose gel electrophoresis was performed and the DNA fragment containing ori (2.14
Kbp) is separated and purified to obtain DNA as an ethanol precipitate,
The precipitate was dissolved in 9.0 t of 10 t / 0.1E (-1 g / l).

Then, the BamHI / XhoI fragment containing the cos and ori of pDcos Tc ^r a DNA solution 1μl (~1μg), the pBR-327 · XhoI ^p
DNA solution of BamHI / XhoI fragment containing ori 1 μl (~ 1 μg)
Were ligated by reacting 20 units of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

Then, after transforming E. coli (HB101) with 10 μl of this DNA solution, a Tc ^r clone was obtained. Then examine the characteristics of plasmid DNA possessed by these clones, two cos and two ori, plasmid has a Tc ^r: p
It was obtained Dcos Tc ^r (2ori) E. coli with (Figure 7).

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Tc ^r (2or
The plasmid DNA of i) was prepared.

(C) Construction and preparation of pSV2-dhfrΔ (BglII) pSV2-dhfr [Subramani, Mulligan & Berg, Mol. Cell.
Biol., 1 , 854 (1981)] 10 μg in 100 μl of reaction mixture,
It was digested by reacting with 8.5 unit BglII at 37 ° C for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1E 7 µl (~ 1 µg / µ
l).

Next, 1 μl of the DNA solution digested with this BglII (up to 1 μl
g) in a reaction mixture of 25 μl with 2 units of Klenow enzyme
The reaction was performed at 22 ° C for 30 minutes, and the cohesive end was changed to blunt end. Then heat the reaction mixture at 65 ° C for 10 minutes and heat it to Klenow.
The enzyme was inactivated.

Next, add 10 μl (~ 0.4 μg) of this DNA solution to the reaction mixture 50
After cyclization with 15 μl of T4-DNA ligase (15 units) at 12 ° C. for 17.5 hours, folloWed by phenol extraction, the DNA Was obtained as an ethanol precipitate.
It was dissolved in 20 μl of HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Then, the characteristics of the plasmid DNA of these clones were examined to find a plasmid lacking the BglII site of pSV2-dhfr:
Escherichia coli having pSV2-dhfrΔ (BglII) (Fig. 7) was obtained.

This E. coli was grown in TYG-P medium 1, and ccc-DNA was separated and purified from the cells to obtain pSV2-dhfrΔ (Bg
lII) plasmid DNA was prepared.

(D) Preparation and preparation of pDcos Tc ^r / ori 20 μg of pDcos Tc ^r (2ori) was added to the reaction mixture 500 μl [60 mM N
aCl, 6mM Tris-HCl (pH7.5 ), 6mM MgCl 2, 6mM DTT, 100
μg / ml BSA] with PvuII (42.5 units) and PstI (35.8 units) at 37 ° C. for 2 hours for complete digestion, followed by 0.8% preparative agarose gel electrophoresis.
Number of cos and ori, was separated and purified DNA fragment (3.1 kbp) containing the Tc ^r, to obtain a DNA as ethanol precipitation, the precipitate 10t / 0.1
It was dissolved in 9.5 μl of E (˜1 μg / μl).

On the other hand, add 20 μg of pSV2-dhfrΔ (BglII) to the reaction mixture 500 μm.
1 with 38.8 unit PvuII and 32.7 unit PstI and 3
After 2 hours of reaction at 7 ℃ for complete digestion, 0.8% preparative agarose gel electrophoresis was performed to obtain DN containing SV-dhfr.
The A fragment (2.45 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 7 μl of 10 t / 0.1E (˜1 μg / μl).

Then, P including cos and ori, Tc ^r of pDcos Tc ^r (2ori)
1 μl (~ 1 μg) DNA solution of vuII / PstI fragment and pSV2-
DNA of PvuII / PstI fragment containing SV-dhfr of dhfrΔ (BglII)
1 μl of the solution (˜1 μg) was ligated by reacting 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was extracted with 0.1 M Tris-HCl (pH 7.5).
It was dissolved in 20 μl of the solution.

Then, after transforming E. coli (HB101) with 10 μl of this DNA solution, a Tc ^r clone was obtained. Then, by examining the characteristics of plasmid DNA possessed by these clones, two cos and ori, Tc ^r, plasmids having a ^{SV-dhfr: pDcos Tc r /} ori ( 4th and 7th Figure) to give E. coli with . In addition, this strain has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, as “Microindustrial Research Institute No. 7554 (FERMD-7554)”.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells, and pDcos Tc ^r / ori
Was prepared.

Example 3 Preparation and Preparation of pDcos Ap ^r / ori (FIG. 8) (a) Preparation and Preparation of pBR-327 XhoI ^a 10 μg of pBR-327 in 100 μl of the reaction mixture was added to 110 units of 110 units.
It was digested by reacting with AvaI at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 7 µl (~ 1 µg / µl).

Next, 1 μl of DNA solution digested with this AvaI (up to 1 μl
g) in a reaction mixture of 25 μl with 2 units of Klenow enzyme
The reaction was performed at 22 ° C for 30 minutes, and the cohesive end was changed to blunt end. Then, heat the reaction solution at 65 ° C. for 10 minutes and then use Klen.
ow enzyme inactivated 10 μl (0.4 μg) of this DNA solution and XhoI linker [d
2 μg of (pC-C-T-C-G-A-G-G)] was ligated with 20 μl of the reaction mixture by reacting with 6 units of T4-DNA ligase at 22 ° C. for 6 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was treated with 600 μl of the reaction mixture to obtain 1000 units of
After digestion by reacting with XhoI for 4 hours at 37 ° C., it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify the 3.27 Kbp DNA fragment, and the DNA was obtained by ethanol precipitation.

This DNA precipitate was mixed with 20 µl of the reaction mixture to prepare 6 units of T4-DNA.
After cyclization by reacting with ligase at 12 ° C for 17.5 hours,
A DNA was obtained as an ethanol precipitate by phenol extraction, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^r clones were obtained. Then, by examining the characteristics of plasmid DNA possessed by these clones, the plasmid was inserted the XhoI linker AvaI site of pBR-327: pBR-327 · XhoI a ( Figure 8) to give E. coli with.

After growing this E. coli in TYG-P medium 1,
The ccc-DNA from the cells was separated and purified, and plasmid DNA was prepared in pBR-327 · XhoI ^a.

(B) Preparation and preparation of pDcos Ap ^r / ori / dhfrΔ (BglII) 20 μg of pDcos Tc ^r / ori was reacted with 200 μl of the reaction mixture for 2 hours at 37 ° C. with 106 units of EcoRI, and then digested with 500 μl of the reaction mixture for 530 It was digested by reacting with XhoI of unit at 37 ° C. for 2 hours. Then, it was subjected to 0.8% preparative agarose gel electrophoresis and the DNA containing two cos and SV-dhfr.
The fragment (~ 2.9 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 7 µl of 10t / 0.1E (~ 1 µg / µl).

On the other hand, 20 μg of pBR-327 / XhoI ^{a was} added to 200 μl of the reaction mixture to prepare 1
After digesting with 67 units of EcoRI for 2 hours at 37 ℃, 500 μl of the reaction mixture was added to 880 units of XhoI and 37
Digested at ℃ for 2 hours. Then, it was subjected to 0.8% preparative agarose gel electrophoresis, and a DNA fragment containing ori and Ap ^r (1.8
4 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 8 μl of 10 t / 0.1E (˜1 μg / μl).

And EcoR including pDcos Tc ^r / ori cos and SV-dhfr
1 μl (~ 1 μg) DNA solution of I / XhoI fragment and pBR-327
・ DNA solution 1 of EcoRI / XhoI fragment containing ori and Ap ^r of XhoI ^a
μl (~ 1 μg) and 20 μl of reaction mixture
Ligation was performed by reacting with T4-DNA ligase at 12 ° C for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate. The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution 2
It was dissolved in 0 μl.

Next, 10 μl of this DNA solution was transformed with Escherichia coli (HB101) to obtain Ap ^r clone. Then, the characteristics of the plasmid DNA of these clones were examined to find a plasmid having two cos and ori, Ap ^r , SV-dhfrΔ (BglII): pDcos Ap ^r / ori / dhfrΔ (BglII) (8th
E. coli having (Fig.) Was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / ori /
A dhfrΔ (BglII) plasmid DNA was prepared.

(C) Preparation and preparation of pSV2-dhfrΔ (BamHI / BglII) 10 μg of pSV2-dhfr was reacted with 48.5 units of BglII in 100 μl of reaction mixture at 37 ° C. for 2 hours for digestion, and further with 250 μl of reaction mixture, 58.2 units of BamHI. And reacted for 2 hours for digestion. And after deproteinizing with phenol extraction,
The DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1E 7 μl
(˜1 μg / μl).

Next, 1 μl of this DNA solution digested with BglII and BamHI
(~ 1 μg) in 25 μl of reaction mixture, 2 units of Klen
blunt cohesive end by reacting with ow enzyme for 30 minutes at 22 ℃
I changed it to end. Then, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme.

Next, add 10 μl (~ 0.4 μg) of this DNA solution to the reaction mixture 50
After cyclization with 15 μl of T4-DNA ligase (15 units) at 12 ° C. for 17.5 hours, folloWed by phenol extraction, the DNA Was obtained as an ethanol precipitate.
It was dissolved in 20 μl of HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Next, the characteristics of the plasmid DNA possessed by these clones were examined, and the DN between the BglII site and the BamHI site of pSV2-dhfr was examined.
Plasmid lacking A fragment (0.85 Kbp): pSV2-dhfrΔ (B
Escherichia coli having amHI / BglII) (Fig. 8) was obtained.

After growing this E. coli in TYG-P medium 1,
Ccc-DNA was separated and purified from the cells and pSV2-dhfrΔ (BamH
I / BglII) plasmid DNA was prepared.

(D) Preparation and preparation of pDcos Ap ^r / ori / dhfrΔ (BamHI / BglII) 20 μg of pDcos Ap ^r / ori / dhfrΔ (BglII) was added to the reaction mixture 200
μl was used to react with 2.9 units of PstI at 37 ° C. for 30 minutes for limited digestion. Next, deproteinize by phenol extraction, then DNA
Was obtained as an ethanol precipitate.

This DNA precipitate was mixed with 200 μl of the reaction mixture to obtain 34.0 units of PvuI.
It was digested by reacting with I at 37 ° C. for 2 hours. Then, it was subjected to 0.8% preparative agarose gel electrophoresis, and two cos and o
The DNA fragment (2.3 Kbp) containing ri and Ap ^r was separated and purified to obtain the DNA as an ethanol precipitate. The precipitate was 10 t / 0.1E 7 μl (~ 1
μg / μl).

On the other hand, 20 μg of pSV2-dhfrΔ (BamHI / BglII) was mixed with 500 μl of the reaction mixture to obtain 46.8 units of PvuII and 39.5 units of PvuII.
After reacting with PstI at 37 ℃ for 2 hours to completely digest it,
SV-dhfr was subjected to 8% preparative agarose gel electrophoresis.
A DNA fragment (1.61 Kb) containing DNA was isolated and purified to obtain DNA as an ethanol precipitate.
μl).

Then, the cos and or of pDcos Ap ^r / ori / dhfrΔ (BglII)
1 μl of DNA solution of PvuII / PstI fragment containing i and Ap ^r (~ 1 μm
g) and 1 μl (to 1 μg) of a DNA solution of a PvuII / PstI fragment containing SV-dhfr of pSV2-dhfrΔ (BamHI / BglII) in a reaction mixture of 20 μl and 6 units of T4-DNA ligase at 12 ° C.
The reaction was carried out for 7.5 hours to ligate. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.
It was dissolved in 20 μl of Tris-HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r clone was obtained. Next, the characteristics of the plasmid DNA possessed by these clones were examined, and two cos and ori, Ap ^r , SV-dhfrΔ (BamHI / BglI
I) -containing plasmid: pDcos Ap ^r / ori / dhfrΔ (BamHI /
BglII) (FIG. 8) was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells, and pDcos Ap ^r / ori /
A plasmid DNA of dhfrΔ (BamHI / BglII) was prepared.

^{(E) pDcos Ap r / ori} / Create dhfrΔ (BamHI) and Preparation pDcos Ap ^r / ori / dhfrΔ the (BamHI / BglII) 10μg, the reaction mixture 100 [mu] l, was reacted for 2 hours with EcoRI and 37 ° C. of 73.7 Yunitsuto Digested. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was extracted with 10 t / 0.1E 7
It was dissolved in μl (˜1 μg / μl).

Next, 1 μl of this DNA solution digested with EcoRI (up to 1 μl
g) in a reaction mixture of 25 μl with 2 units of Klenow enzyme
The reaction was performed at 22 ° C for 30 minutes, and the cohesive end was changed to blunt end. Then, heat the reaction solution at 65 ° C. for 10 minutes and then use Klen.
ow The enzyme is inactivated.

10 .mu.l (0.4 .mu.g) of this DNA solution and 2 .mu.g of BamHI linker [d (pC-G-G-A-T-C-C-G)] in 20 .mu.l of reaction mixture were mixed with 6 units of T4-DNA ligase and 22.degree.
Was reacted for 6 hours and ligated. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was mixed with 600 μl of the reaction mixture to obtain 1000 units of Ba.
After digestion by reacting with mHI at 37 ° C. for 4 hours, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify the 3.91 Kbp DNA fragment, and the DNA was obtained by ethanol precipitation.

This DNA precipitate was mixed with 20 µl of the reaction mixture to prepare 6 units of T4-DNA.
After cyclization by reacting with ligase at 12 ° C for 17.5 hours,
A DNA was obtained as an ethanol precipitate by phenol extraction, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Next, the characteristics of plasmid DNA possessed by these clones were examined, and EcoRI of pDcos Ap ^r / ori / dhfrΔ (BamHI / BglII) was examined.
A plasmid having a BamHI linker inserted into the site (in this case, EcoRI sites are reconstituted on both sides of the BamHI site): pDcos Ap ^r / ori / dhfr (BamHI) (Fig. 8) was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells, and pDcos Ap ^r / ori /
A dhfr (BamHI) plasmid DNA was prepared.

(F) pSVO10 / neo ^r of creation and preparation pAO43 (Oka, Sugisaki & Takanami, J.Mol, Biol, 147, 2
14 (1981)) 20 μg reaction mixture 200 μl (60 mM NaCl, 6 mM
Tris-HCl (pH8.0), 10 mM MgCl ₂ , 6 mM DTT, 100 μg / ml B
SA), a 150-unit restriction enzyme AvaII (New England Bi
(Olabs) and digest at 37 ℃ for 2 hours,
The DNA fragment containing neo ^r (1.24 Kbp) was separated and purified by 0.8% preparative agarose gel electrophoresis to obtain DNA as an ethanol precipitate, and the precipitate was 10 t / 0.1E 5 μl (~ 1 μg / μ).
l).

2 μl (~ 2 μg) of this DNA solution, 50 μl of reaction mixture
Then, it was reacted with 4 units of Klenow enzyme at 22 ° C. for 30 minutes to change the cohesive end to blunt end. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 1.5 μl (˜1 μg / μl).

On the other hand, pSVO10 〔{πVX 〔Brain seed, Nucleic Acids R
es., 11 , 2427 (1983)] with polylinker pSV
O1 〔Tjan, R, et al, Proc, Natl, Acad.Sci.USA, 77 , 6491
(1980)] related plasmid}] 10 μg in a reaction mixture 10
After digestion with 0 µl of BamHI of 129 units for 2 hours at 37 ° C, phenol extraction was performed to obtain DNA as ethanol precipitate. The precipitate was 10 t / 0.1E 7 µl (~ 1 µg / µl).
l).

2 μl (~ 2 μg) of this DNA solution, 50 μl of reaction mixture
Then, it was reacted with 4 units of Klenow enzyme at 22 ° C. for 30 minutes to change the cohesive end to blunt end. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 1.5 μl (˜1 μg / μl).

Next, 1 μl (~ 1 μg) of a DNA solution obtained by treating a DNA fragment containing neo ^r of pAO43 with Klenow enzyme and 1 μl (~ 1 μg of a DNA solution obtained by digesting pSVO10 with BamHI and treating with Klenow enzyme).
g) was ligated by reacting 20 μl of the reaction solution with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as ethanol precipitate,
The precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Km ^r (neo ^r ) clones were obtained. Then a plasmid carrying these clones
The characteristics of the DNA were examined to obtain Escherichia coli harboring a plasmid: pSVO10 / neo ^r (Fig. 8) in which neo ^r was inserted into the BamHI site of pSVO10.

The E. coli, in TYG-P medium 1, then grown, the ccc-DNA from the cells was separated and purified, and plasmid DNA was prepared for pSVO10 / neo ^r.

(G) pBR-322 / neo r creation and preparation pSVO10 / neo ^r 20μg an after digestion by 2 hours with PstI and 37 ° C. 46.6 Yunitsuto reaction mixture 200 [mu] l, and EcoRI further reaction mixture 500μl in 166 Yunitsuto It was digested by reacting at 37 ° C for 2 hours. Then, it was subjected to 0.8% preparative agarose gel electrophoresis, and the DNA fragment containing neo ^r (1.24 Kb
p) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 5 μl of 10 t / 0.1E (˜1 μg / μl).

On the other hand, 20 µg of pBR-322 was digested with 200 µl of the reaction mixture by reacting with 37.2 units of PstI at 37 ° C for 2 hours and then further reacted with 500 µl of the reaction mixture with 132 units of EcoRI for 2 hours. Thereafter, agarose gel electrophoresis 0.8% preparation, DNA fragment containing ori and Tc ^r (3.6 Kb
p) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 10 t / 0.1E 11 μl (˜1 μg / μl).

Then, D of EcoRI / PstI fragment containing the neo ^r of PSVO10 / neo ^r
NA solution and 1μl (~1μg), and pBR-322 of ori and DNA solution 1 [mu] l of EcoRI / PstI fragment containing the Tc ^r (~1μg), and T4-DNA ligase reaction mixture 20μl 6 Yunitsuto 1
The reaction was carried out at 2 ° C for 17.5 hours for ligation. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Km ^r (neo ^r ) and Tc ^r , Ap ^{s were obtained.}
I got a clone. Then, the properties of the plasmid DNA having these clones were examined to obtain Escherichia coli having the plasmid: pBR-322 / neo ^r (Fig. 8) in which the neo ^r fragment was inserted into pBR-322.

The E. coli, in TYG-P medium 1, then grown, and plasmid DNA was prepared in pBR-322 / neo ^r a ccc-DNA from the cells was separated and purified.

(H) Preparation and preparation of pBR-322 / neo ^r Δ (EcoRI / BalI) 10 µg of pBR-322 / neo ^r was reacted with 40 units of BalI in 100 µl of the reaction mixture at 37 ° C for 2 hours for digestion, followed by further reaction. 250 μl of the mixed solution was reacted with 120 units of EcoRI at 37 ° C. for 2 hours for digestion. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was extracted with 10t / 0.1E.
It was dissolved in 7 μl (˜1 μg / μl).

Next, 1 μl of the DNA solution digested with this BalI and EcoRI (~
1 μg) with 25 μl of the reaction mixture and reacted with 2 units of Klenow enzyme at 22 ° C for 30 minutes to blunt en the cohesive end.
changed to d. Then, heat the reaction solution at 65 ° C for 10 minutes to obtain K
deactivated the lenow enzyme.

Next, add 10 μl (~ 0.4 μg) of this DNA solution to the reaction mixture 50
After cyclization with 15 μl of 15-unit T4-DNA ligase at 12 ° C for 17.5 hours (in this case, EcoRI site is reconstituted), phenol extraction was performed and DN extraction was performed.
A was obtained as an ethanol precipitate, and the precipitate was mixed with 0.1 M Tris-HCl (p
H7.5) solution was dissolved in 20 μl.

After transforming E. coli (HB101) with 10 μl of this DNA solution, neo ^r (Km ^r ) and Tc ^s , Ap were obtained.
^s clone was obtained. Then examine the characteristics of plasmid DNA possessed by these clones, pBR-322 / neo ^r of EcoRI sites and DNA fragment between the BalI site (1.42Kbp) the lack ^{plasmid: pBR-322 / neo r Δ} (EcoRI / BalI) (Fig. 8) was obtained.

The E. coli, in TYG-P medium 1, then grown, and separated and purified ccc-DNA from the cells, pBR-322 / neo ^r
A Δ (EcoRI / BalI) plasmid DNA was prepared.

(I) pDcos Ap ^r / ori creation and preparation ^{pDcos Ap r / ori / dhfr (} BamHI) 20μg reaction mixture 200μ
The reaction was carried out for 30 minutes at 37.degree. C. with 4.1 units of PstI for limited digestion. Then, after deproteinizing by phenol extraction, DNA was obtained as an ethanol precipitate.

This DNA precipitate was mixed with 200 μl of the reaction mixture to obtain 49 units of PvuI.
It was digested by reacting with I at 37 ° C. for 2 hours.

Then, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify the DNA fragment (2.3 Kbp) containing the two cos, ori, and Ap ^r, and the DNA was obtained as an ethanol precipitate.
It was dissolved in 8 μl of 0t / 0.1E (˜1 μg / μl).

On the other hand, the reaction mixture 500μl of ^{pBR-322 / neo r Δ (} EcoRI / BalI) 20μg, 56 Yunitsuto PvuII and 47 Yunitsuto of Ps
After reacting with tI at 37 ℃ for 2 hours to completely digest, 0.8
% Preparative agarose gel electrophoresis, including neo ^r
The DNA fragment (1.9 Kbp) was separated and purified to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 8 μl of 10 t / 0.1E (˜1 μg / μl).

And pDcos Ap ^r / ori / dhfr (BamHI) cos and ori,
1 μl (~ 1 μg) DNA solution of PvuII / PstI fragment containing Ap ^r
And PvuII containing neo ^r of pBR-322 / neo ^r Δ (EcoRI / BalI)
/ PstI fragment DNA solution 1 μl (~ 1 μg) and reaction mixture
Ligation was performed by reacting with 20 μl of 6-unit T4-DNA ligase at 12 ° C. for 17.5 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was mixed with 0.1 M Tris-HC.
It was dissolved in 20 μl of 1 (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and neo ^r (Km ^r ) clones were obtained. Next, plasmid DNA containing these clones
Was examined to obtain Escherichia coli harboring a plasmid having two cos and ori, Ap ^r and neo ^r : pDcos Ap ^r / ori (Fig. 1, 8). This strain has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as "Microindustrial Research Institute No. 7553 (FERMP-7553)".

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / ori
Was prepared.

Example 4 Preparation and preparation of pDcos Ap ^r / ori / gpt / TK (No. 9)
(A) Preparation and preparation of pSV2-gptΔ (HindIII / BglII) pSV2-gpt [Richard, Mulligan & Berg, Mol.Cell.Bio
l., 1 , 449, (1981)] 10 μg in 100 μl of the reaction mixture,
It was digested by reacting with HindIII of unit and BglII of 46 unit at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was extracted with 10t / 0.1E.
It was dissolved in 7 μl (˜1 μg / μl).

Next, 1 μl of the DNA solution digested with this HindIII and BglII
1 unit (up to 1 μg) was added to 2 units of Klen in 25 μl of the reaction mixture.
ow the enzyme with ow enzyme for 30 minutes at 22 ℃ and blun the cohesive end.
changed to t end. Then, the reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the Klenow enzyme.

Next, add 10 μl (~ 4.9 μg) of this DNA solution to the reaction mixture 50
After cyclization with 15 μl of T4-DNA ligase (15 units) at 12 ° C. for 17.5 hours, folloWed by phenol extraction, the DNA Was obtained as an ethanol precipitate.
It was dissolved in 20 μl of HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and a plasmid lacking the DNA fragment (0.12 Kbp) between the HindIII site and the BglII site of pSV2-gpt: pSV2-gpt
Escherichia coli having Δ (HindIII / BglII) (FIG. 9) was obtained.

This E. coli was grown in TYG-P medium 1, and then ccc-DNA was separated and purified from the cells to obtain pSV2-gptΔ (Hin
dIII / BglII) plasmid DNA was prepared.

(B) Preparation and preparation of pSV2-gptΔ (HindIII / BglII) / BamHI pSV2-gptΔ (HindIII / BglII) 10 μg was added to the reaction mixture 100
This was digested with 1 μl of PvuII (18.8 unit) at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10 t / 0.1E 7 μl (~ 1
μg / μl).

Next, 0.4 μl (-0.4 μg) of this DNA solution and 2 μm of BamHI linker [d (pC-A-G-A-T-C-C-G)]
20 g of the reaction mixture was reacted with 6 units of T4-DNA ligase at 22 ° C for 6 hours for ligation. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate.

This DNA precipitate was mixed with 600 μl of the reaction mixture to obtain 1000 units of Ba.
After digestion with mHI for 4 hours at 37 ℃, it was electrophoresed on 0.8% preparative agarose gel to give a DN of 5.1 Kbp in length.
The A fragment was separated and purified, and the DNA was subjected to ethanol precipitation.

This DNA precipitate was mixed with 20 µl of the reaction mixture to prepare 6 units of T4-DNA.
After cyclization by reacting with ligase at 12 ° C. for 17.5 hours, phenol extraction was carried out to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. Next, the characteristics of the plasmid DNA containing these clones were examined, and a Bv site at the PvuII site of pSV2-gptΔ (HindIII / BglII) was examined.
Plasmid with amHI linker inserted: pSV2-gptΔ (Hi
E. coli having ndIII / BglII) / BamHI (Fig. 9) was obtained.

After this E. coli was grown in TYG-P medium 1, pccV2-gptΔ (Hin
A plasmid DNA of dIII / BglII) / BamHI was prepared.

(C) Preparation and preparation of pDcos Ap ^r / ori / gpt (2 BamHI) 20 μg of pSV2-gptΔ (HindIII / BglII) BamHI was reacted with 200 μl of the reaction mixture for 2 hours at 37 ° C. with BamHI of 226 units, followed by digestion. , 0.8% preparative agarose gel electrophoresis to separate and purify the DNA fragment containing gpt (2.2 Kbp) to obtain DNA as an ethanol precipitate. The precipitate was 10 t / 0.1E.
It was dissolved in 8.5 μl (˜1 μg / μl).

On the other hand, the ^{pDcosAp r / ori / dhfr (BamHI} ) 10μg reaction mixture
It was digested by reacting with 100 μl of 74 unit BamHI at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1 E7 μl (~ 1
μg / μl).

Then, pSV2-gptΔ (HindIII / BglII) / BamHI gpt
Containing 1 μl (~ 1 μg) of BamHI fragment DNA solution containing pDco
sAp ^r / ori / dhfr DNA solution 1μ the (BamHI) was digested with BamHI
1 unit (~ 1 μg) with 20 μl of reaction mixture and 6 units of T4
-Ligation was carried out by reacting with DNA ligase at 12 ° C for 17.5 hours.
Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 20 μl of 0.1 M Tris-HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r clone was obtained. Next, the characteristics of the plasmid DNA possessed by these clones were examined, and a plasmid in which gpt was inserted into the BamHI site of pDcos Ap ^r / ori / dhfr: pDcos Ap ^r / ori / gpt (2BamHI) (9th
E. coli having (Fig.) Was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / ori / gp.
Plasmid DNA of t (2BamHI) was prepared.

(D) the pDcos Ap ^r / ori / gpt creation and preparation ^{pDcos Ap r / ori / gpt (} 2BamHI) 10μg, the reaction mixture 100
It was reacted with 9.1 unit of BamHI in μl for 30 minutes at 37 ° C. for limited digestion. After deproteinization with phenol extraction, DNA
Was obtained as an ethanol precipitate, and the precipitate was mixed with 10 t / 0.1E 7 μl (~
1 μg / μl).

Next, 1 μl of DNA solution (~ 1
μg) with 2 units of Klenow enzyme in 25 μl of reaction mixture
After reacting at 22 ° C for 30 minutes, changing cohesive end to blunt end, 0.8% preparative agarose gel electrophoresis was performed, and a 6.3 Kbp-long DNA fragment was separated and purified to obtain DNA as an ethanol precipitate. .

This DNA precipitate was mixed with 20 µl of the reaction mixture to prepare 6 units of T4-DNA.
After cyclization by reacting with ligase at 12 ° C for 17.5 hours,
Extraction with phenol was performed to obtain DNA as an ethanol precipitate. The precipitate was dissolved in 0.1 M Tris-HCl (pH 7.5) solution (20 μl).
Dissolved in.

After transforming E. coli (HB101) with 10 μl of this DNA solution, an Ap ^r clone was obtained. We next characterized the plasmid DNAs carrying these clones to find the B between the cos and gpt of pDcos Ap ^r / ori / gpt (2BamHI).
Escherichia coli having a plasmid lacking amHI site: pDcos Ap ^r / ori / gpt (Fig. 9) was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / ori /
A gpt plasmid DNA was prepared.

(E) Preparation and preparation of pTK-4 / BamHI pTK-4 [Ishiura, M., et al. Mol. Cell Biol, 2 , 607 (19
82)] 10 μg was added to 100 μl of the reaction mixture to obtain 3.0 units of PvuI.
After reacting with I for 30 minutes at 37 ° C and limited digestion, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify a 6.43 Kbp long DNA fragment to obtain DNA as an ethanol precipitate. It was dissolved in 3 μl of 0.1E (˜1 μg / μl).

Next, 0.4 μl (-0.4 μg) of this DNA solution and 2 μm of BamHI linker [d (pC-G-G-A-T-C-C-G)]
20 g of the reaction mixture was reacted with 6 units of T4-DNA ligase at 22 ° C for 6 hours for ligation.

Then, 10 μl of this reaction mixture was reacted with 15 units of T4-DNA ligase in 50 μl of the reaction mixture at 12 ° C. for 17.5 hours for cyclization. After that, phenol extraction was performed to obtain DNA as an ethanol precipitate, which was precipitated with 0.1 M Tris-HCl (pH 7.
5) The solution was dissolved in 20 μl.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^r clones were obtained. Then, the characteristics of the plasmid DNA of these clones were examined, and a plasmid having a BamHI linker inserted at the PvuII site between ori and TK of pTK-4: pTK-4 / BamHI
E. coli having (Fig. 9) was obtained.

This Escherichia coli was grown in TYG-P medium 1, and ccc-DNA was separated and purified from the cells to prepare pTK-4 / BamHI plasmid DNA.

(F) Preparation and preparation of pTK-4 / BamHI / BamHI 10 μg of pTK-4 / BamHI was reacted with 9 units of BamHI in 100 μl of the reaction mixture at 37 ° C. for 30 minutes for limited digestion,
A phenol extraction was performed to obtain DNA as an ethanol precipitate.

This ethanol precipitate was completely digested by reacting 100 μl of the reaction mixture with 15 units of PvuII at 37 ° C. for 2 hours, followed by phenol extraction to obtain DNA as ethanol precipitate, and the precipitate was 10 t / 0.1E 3 μl (~ 1 μg / ~ 1 μg / μl).

Next, 1 μl (~ 1 μg) of the DNA solution digested with this BamHI and further digested with PvuII was added to 25 μl of the reaction mixture to prepare 2
Incubate with Klenow enzyme from unit for 30 minutes at 22 ℃ to remove cohe
After changing the sive end to blunt end, it was subjected to 0.8% preparative agarose gel electrophoresis to separate and purify the DNA fragment (4.67 Kbp) containing ori and Ap ^r , TK to obtain the DNA as ethanol precipitation.

This DNA precipitate was mixed with 20 µl of the reaction mixture to prepare 6 units of T4-DNA.
After cyclization by reacting with ligase at 12 ° C for 17.5 hours (in this case, BamHI site is reconstituted), phenol extraction was performed to obtain DNA as ethanol precipitate, and the precipitate was extracted with 0.1M Tris-HCl (pH7.5 ) Dissolved in 20 μl of solution.

Then, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r and Tc ^s clones were obtained. Then examine the characteristics of plasmid DNA possessed by these clones, pTK-4 / BamHI of PvuII and BamHI sites (Tc ^r on) DNA fragment (1.76Kbp) the lack plasmid between: pTK-4 / BamHI / E. coli with BamHI (Fig. 9) was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells and pTK-4 / BamHI / B was isolated.
AmHI plasmid DNA was prepared.

(G) Preparation and preparation of pDcos Ap ^r / ori / gpt / TK (2BamHI) pTK-4 / BamHI / BamHI 20 μg was added to the reaction mixture 200 μl in 247
After reacting with unit's BamHI at 37 ℃ for 2 hours and digesting, it was subjected to 1.0% preparative agarose gel electrophoresis and TK
A DNA fragment (2 Kbp) containing DNA was isolated and purified to obtain DNA as an ethanol precipitate. The precipitate was mixed with 10 t / 0.1E 6 μl (∼1 μg / μl).
l).

On the other hand, pDcos Ap ^r / ori / gpt 10 μg was added to the reaction mixture 100 μl
Then, it was digested by reacting with 91 unit BamHI at 37 ° C for 2 hours. After deproteinization by phenol extraction, DNA was obtained as ethanol precipitate, and the precipitate was 10t / 0.1E 7 μl (~ 1 μg / μ
l).

Then, D of BamHI fragment containing TK of pTK-4 / BamHI / BamHI
Bam the NA solution 1 μl (~ 1 μg) and pDcos Ap ^r / ori / gpt
1 μl (~ 1 μg) of the DNA solution digested with HI was added to 20 μl of the reaction mixture and 6 units of T4-DNA ligase and 17.5 at 12 ° C.
After reacting for a period of time, they were connected. After that, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was mixed with 0.1 M Tris-
It was dissolved in 20 μl of HCl (pH 7.5) solution.

Next, after transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r clone was obtained. Then, by examining the characteristics of plasmid DNA possessed by these clones, pDcos Ap ^r / ori / gpt plasmid was inserted the TK into the BamHI ^{site: pDcos Ap r / ori / gpt} / TK (2BamHI) ( 9
E. coli having (Fig.) Was obtained.

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells to obtain pDcos Ap ^r / ori /
A plasmid DNA of gpt / TK (2BamHI) was prepared.

^{(H) pDcos Ap r / ori} / gpt / TK creation and preparation ^{pDcos Ap r / ori / gpt /} TK (2BamHI) 10μg reaction mixture 10
This was digested with 0 μl of BamHI (6.9 unit) at 37 ° C. for 30 minutes for limited digestion. After deproteinization with phenol extraction, DN
A was obtained as an ethanol precipitate, and the precipitate was mixed with 10 t / 0.1E 7 μl (~
1 μg / μl).

Next, 1 μl of DNA solution (~ 1
μg) with 2 units of Klenow enzyme in 25 μl of reaction mixture
After reacting at 22 ° C for 30 minutes and changing cohesive end to blunt end, 0.8% preparative agarose gel electrophoresis was performed to separate and purify a DNA fragment with a length of 8.3 Kbp to obtain DNA as an ethanol precipitate. .

The DNA precipitate was reacted with 20 μl of the reaction mixture with 6 units of T4-DNA ligase at 12 ° C. for 17.5 hours for cyclization, followed by phenol extraction to obtain the DNA as ethanol precipitate. The precipitate was 0.1M Tris-HCl. (PH 7.5) solution 20
It was dissolved in μl.

After transforming E. coli (HB101) with 10 μl of this DNA solution, Ap ^r clone was obtained. Then, the characteristics of the plasmid DNA possessed by these clones were examined, and p
Ba between TK and Ap ^{r of} Dcos Ap ^r / ori / gpt / TK (2BamHI)
Plasmid lacking mHI site: pDcosAp ^r / ori / gpt / TK
E. coli having (Figs. 5 and 9) were obtained. In addition, this strain is
It has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as "Micro Engineering Research Institute No. 7552 (FERMP-7552)".

After this E. coli was grown in TYG-P medium 1, ccc-DNA was separated and purified from the cells, and pDcos Ap ^r / ori /
A plasmid DNA of gpt / TK was prepared.

Example 5 Genomic DNA (foreign DNA) by pDcos Ap ^r / ori
(See Fig. 2) (a) Preparation of high molecular weight genomic DNA Recombination of thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) having the nucleotide sequence of pBR-322 mouse L cells (Ltk transfected with body ^- [HSV-
1TK ⁺ ]) was cultured in Eagle MEM supplemented with 10% calf serum, and confluent monolayers (100 mm-9 plates,
~ 10 ⁷ cells / plate, PBS (137mM NaCl1,3mM KCl
After washing twice with 1,8mM Na ₂ HPO ₄ , 1mM KH ₂ PO ₄ , solubilizer (100μg / ml Proteinase K 0.5% SDS, 150mM NaCl, 10mM
EDTA, 10mM Tris-HCl (pH7.5)) 1.0ml per plate
In addition, it was solubilized by incubating at 65 ° C for 15 to 30 minutes. The solubilized product was transferred to a centrifuge tube having a volume of 50 ml, further incubated at 37 ° C. overnight, and 100 μg / ml of Protinase K was added during the incubation. After the reaction, gently extract twice with an equal volume of phenol saturated with Tris buffer, and the aqueous layer is washed with 50 mM Tris-HCl (pH 8.0), 1
Dialysis was performed 4 times against 3 liters of dialysis buffer consisting of 0 mM EDTA and 10 mM NaCl. The dialysate is 50 μg / ml DNase-free.
RNase A was added and the mixture was kept warm at 37 ° C for 3 hours to decompose RNA.
After the reaction, gently extract with an equal volume of phenol: chloroform mixture twice, and extract the aqueous layer with 10 mM Tris-HCl (pH 8.0), 1 mM.
It was dialyzed 9 times against 3 l of an aqueous solution (TE) consisting of EDTA to obtain 50 ml of a purified DNA solution. The absorbance (OD) of the DNA solution at 260 nm and 280 nm is 0.667, 0.363 (OD ₂₈₀ / OD ₂₈₀ /
OD ₂₆₀ = 0.544) and the DNA concentration is 0.667 × 0.050 (μg
/1OD)×10=0.334 μg / μl.

(B) Preparation of 35 to 45 Kbp genomic DNA 5.98 ml (2 mg) of the purified genomic DNA solution was added to the reaction mixture 2
9.96 ml (50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgC
l ₂ , 100 μg / ml BSA) 15.6 unit Sau 3A (New Engl
and Biolabs) for 1 hour at 37 ° C. for limited digestion, 1.3 ml of 0.5 M EDTA (pH 8.0) solution was added, and the mixture was gently extracted twice with an equal volume of Tris buffer saturated phenol. The aqueous layer was concentrated to 10 ml with secondary butanol, then 1.1 ml of 3.0 M sodium acetate and 20 ml of ethanol were added.
l was added to obtain ethanol-precipitated genomic DNA, which was washed with 70% ethanol and dried with a water-jet pump to dissolve the ethanol-precipitate in 2 ml of TE.

Next, 300 μl of this DNA solution was subjected to 5-25% sucrose density gradient centrifugation [20 mM Tris-HCl (pH 7.6), 5 mM EDTA, Beckmann SW28, 20,000 rpm, 9 hours, 20 ° C.] to fractionate every 30 drops. did. 20 μl of each fraction was subjected to 0.4% agarose gel electrophoresis to measure the length, DNA fractions having a size of 35 to 45 Kbp were collected, and then dialyzed against TE3l three times. Then, the dialysate was extracted with phenol and the aqueous layer was concentrated with secondary butanol to obtain DNA as an ethanol precipitate,
The precipitate was dissolved in 1 ml of TE to obtain a 35-45 Kbp genomic DNA solution. The OD ₂₆₀ and OD ₂₈₀ of the DNA solution were 0.216 and 0.112 (OD ₂₈₀ / OD ₂₆₀ = 0.519), respectively, at a 50-fold dilution, and the DNA concentration was 0.216 × 0.050 (μg / 1OD) × 50 = 0.54 μg / μl. It was

(C) Preparation of cosmid vector DNA 40 μg of pDcos Ap ^r / ori was digested with 152 μl of PvuII in a reaction mixture of 800 μl at 37 ° C. for 2 hours. Then, phenol extraction was performed to obtain DNA as an ethanol precipitate, and the precipitate was dissolved in 100 μl of 10 t / 0.1E.

95 μl of this PvuII-digested DNA solution was added to the reaction mixture 500
μl (50 mM Tris-HCl (pH 9.0), 1 mM MgCl ₂ , 0.1 mM ZnCl
_2, in 1mM Superujimin) of 6 Yunitsuto phosphatase (Calf intestinal alkaline phosphatase, Boehringer,
Mannheim) at 37 ° C for 15 minutes and at 56 ° C for 15 minutes, and then add 6 units of phosphatase.
The fragments of PvuII were inactivated by reacting for 15 minutes and then at 56 ° C for 15 minutes. After that, H ₂ O 400 μl, STE (100 mM Tris
-HCl (pH8.0), 1M NaCl, 10 mM EDTA) 100 μl, 10% S
50 μl of DS was added and the phosphatase was inactivated by incubating at 68 ° C. for 15 minutes. And after deproteinizing with phenol extraction,
The DNA was obtained as an ethanol precipitate, and the precipitate was 10t / 0.1E 30 μl
Dissolved in.

Next, 30 μl of the DNA solution digested with PvuII and inactivated by phosphatase was digested with 500 μl of the reaction mixture by reacting with 152 units of BamHI at 37 ° C. for 2 hours. After deproteinization by phenol extraction, DNA was obtained as an ethanol precipitate, and the precipitate was dissolved in 30 t of 10 t / 0.1E to obtain a cosmid vector DNA solution. OD ₂₆₀ and OD _{280 of} DNA solution
Is 0.046, 0.026 (OD ₂₈₀ / OD ₂₆₀ = 0.
565), and the DNA concentration is 0.046 × 0.050 (μg / 1OD) × 3
00 = 0.690 μg / μl.

(D) Ligation of 35-45 Kbp genomic DNA with cosmid vector DNA 2.61 μl of 35-45 Kbp genomic solution prepared in (b)
(1.4 μg) and the cosmid vector DNA prepared in (c)
Solution 0.86 μl (0.59 μg) and reaction mixture 10 μl for 5.9
Ligation was performed by reacting with unit T4-DNA ligase at 12 ° C for 17.5 hours. After the reaction was completed, the reaction solution was stored at 4 ° C.

(E) Preparation of packaged extract i) Preparation of cell extract obtained by ultrasonic treatment from BHB2690 E. coli BHB2690 was added to NZY medium (1% NZamine, 0.5% yeast extract, 0.5% NaCl, 0.2% MgCl ₂ ·. 6H ₂ O, pH 7.5) After overnight culture in 100 ml, the OD ₆₀₀ was measured and then the first OD was added to 500 ml of NZY medium (in a 2 l flask) that had been pre-heated to 32 ° C.
Bacteria were inoculated so that ₆₀₀ became 0.025, and shaking culture was started at 32 ° C. Then, when the OD ₆₀₀ reached 0.3 (2 hours and 40 minutes after the start of culture), induction of profage was carried out at 45 ° C. for 15 minutes. Then, the culture temperature was changed to 38 to 39 ° C., and the culture was further vigorously shaken for 3 hours (the induction was examined by adding a drop of chloroform to a small amount of the culture solution).

After the culture was completed, the cells were collected by centrifuging at 6,000 rpm for 10 minutes, and the bacterial cells were collected with 20 mM Tris-HCl (pH8.0), 1 mM EDT.
A, 5 mM β-mercaptoethanol was suspended in 3.6 ml of a buffer solution. After sonication (maximum output: 5 seconds × 20 times) at 4 ° C. or lower, centrifugation was performed at 20,000 rpm for 10 minutes (4 ° C.) to obtain ˜3 ml of supernatant.

Next, 0.5 ml of the above-mentioned buffer and packaging buffer (6 mM Tris-HCl (pH 8.0), 50 mM sperdimine, 50 mM putrescine, 20 mM MgCl ₂ , 30 mM ATP, 30 mM β-mercaptoethanol) 0.5 ml in the supernatant 3 ml. After adding, add 15 μl each
It was dispensed into 1.5 ml of Eppendorf tube, immediately frozen in liquid nitrogen, and stored at -70 ° C.

ii) Preparation of cell extract obtained from BHB2688 by freezing and thawing E. coli 2688 was cultured overnight in 100 ml of NZY medium to obtain OD.
After measuring ₆₀₀ , the N that was previously kept warm at 32 ° C
500 ml of ZY medium (in a 2 l flask) was inoculated with the bacteria so that the initial OD ₆₀₀ was 0.025, and shake culture was started at 32 ° C. Then, when the OD ₆₀₀ reached 0.3 (2 hours from the start of culture
After 40 minutes), induction of profage was performed at 45 ° C. for 15 minutes. Then, the culture temperature was changed to 38 to 39 ° C., and the culture was further vigorously shaken for 3 hours (induction test was carried out by adding one drop of chloroform to a small amount of the culture solution).

After the culture was completed, the cells were collected by centrifuging at 6,000 rpm for 10 minutes, and the cells were taken into a sucrose solution (10% sucrose, 50 mM Tri
After suspending in 3 ml of s-HCl (pH8.0), 0.5 ml each was placed in 6 Etpendorf tubes, and a lysozyme solution (2 mg / ml, 0.25M Tris-HCl (pH8.0)) was added to each tube.
25 μl was added (4 ° C.), mixed gently and immediately frozen in liquid nitrogen.

Next, the tube was thawed on ice to melt the extract, and then 25 μl of packaging buffer was added to each tube and mixed. And each extract together, 21,000rpm
The mixture was centrifuged at 4 ° C. for 1 hour to obtain a supernatant, and 10 μl of the supernatant was dispensed into 1.5 ml of Eppendorf tube, immediately frozen in liquid nitrogen, and stored at −70 ° C.

(F) In vitro packaging The cell extract of BHB2690 and BHB2688 stored at -70 ° C was placed on ice, and the extract of BHB2688 that was thawed first was B.
It was added to the extract of HB2690 and gently mixed. When almost all was melted, 2.5 μl (0.5 μg) of the recombinant DNA solution prepared in (d) was added and mixed well, and then allowed to react at room temperature for 1 hour for in vitro packaging.

After completion of the reaction, SM solution (0.58% NaCl, 0.2% MgSO 4 · 7H
1 ml of ₂ O, 50 mM Tris-HCl (pH 7.5) 0.01% gelatin) and 1 drop of chloroform were added and mixed. Then, the Eppendorf tube was placed on a desk centrifuge for 30 seconds to obtain a supernatant as a transduction-charge particle solution.

(G) Transduction and plating 200 μl of an overnight culture of E. coli HB101 in L-broth (0.4% maltose added) was collected on a desk centrifuge and resuspended in 100 μl of 10 mM MgCl ₂ solution. did. Next, to 100 μl of this bacterial cell suspension, 100 μl of a solution of transduction fare particles diluted 10-fold with SM solution was added, and the mixture was incubated at 37 ° C. for 1 hour.
After incubating for 5 minutes for transduction, 1.1 ml of L-broth was further added, and the mixture was cultured at 37 ° C for 45 minutes with shaking. And
100 μl of this cell suspension was applied to 100 mm L-plate containing 20 μg / ml of ampicillin, and the plate was kept at 37 ° C.
After overnight, the resulting colonies are attached with bamboo sticks and
-Grown in broth (containing 20 μg / ml ampicillin).
The number of colonies per plate was 20-100.

(H) Cultivation of transformants and preparation and characterization of their plasmid DNA Ampicillin-resistant colonies (transformants) 20
After culturing the individual pieces in 2 ml of L-broth (containing 20 μg / ml of ampicillin) overnight, 1.5 ml of each culture solution was placed in 1.5 ml of Eppendorf tube and collected on a desk centrifuge. These cells were treated with solution I (50 mM glucose, 25 mM Tris-HCl.
(PH 8.0), 10 mM EDTA, 4 mg / ml lysozyme), and the suspension was resuspended in 100 μl and then left at room temperature for 5 minutes. Next, solution II
(0.2N NaoH, 1% SDS) (200 μl) was added and gently mixed, and the mixture was kept on ice for 5 minutes, and then the solution III [5M potassium acetate (p
H4.8)] Add 150 μl and mix gently, and add 5 on ice.
I left it for a minute. Then, Eppendorf tube was subjected to a desktop centrifuge to obtain a supernatant liquid, and 2 volumes of ethanol was added thereto to obtain a plasmid DNA as an ethanol precipitate.

A small amount of plasmid DNA in each transformant
After dissolving in 0t / 0.1E, size marker DNA (lambda
DNA: 48 Kbp, Lambda DNA digested with HindIII: 22.3 Kb
p, 9.5 Kbp) and 0.4% agarose gel electrophoresis, 20 of the ampicillin resistant colonies
All plasmids range in size from 22.3 Kbp to 48 Kbp
Presence of DNA was observed.

[Brief description of drawings]

Figure 1 shows the new cosmid vector pDcos Ap ^r / ori, and Figure 2 shows the genome using the new cosmid vector of Figure 1.
Shows the preparation of DNA of the gene bank, Figure 3 shows a novel cosmid vector pDcos Tc ^r, Figure 4 shows a novel cosmid vector pDcos Tc ^r / ori, Fig. 5 new cosmid vector pDcos Ap ^r / ori / gpt / TK, Fig. 6 shows how to make pDcos Tc ^r , Fig. 7 shows how to make pDcos Tc ^r / ori, Fig. 8 shows how to make pDcos Ap ^r / ori, FIG. 9 shows a method for creating pDcos Ap ^r / ori / gpt / TK.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Yoshio Okada 2-22-28 Hanmachi, Minoh City, Osaka Prefecture (56) References Gene, 26, [1983] p. 137-146

Claims (5)

[Claims] 1. The following having the same directionality: cos: Lambda phage cos ori: Origin of replication Ap ^r : Ampicillin resistance expressing region neo ^r : Neomycin resistance expressing cosmid vector pDcosAp ^r / ori and the following cos: cos ori of lambda phage: origin of replication Tc ^r : cosmid vector pDcos Tc ^r shown in the region expressing tetracycline resistance and cos: cos ori of lambda phage: origin of replication Tc ^r : region expressing tetracycline resistance SV-dhfr: SV-40 cosmid vector pDcos Tc ^r / ori shown in the region expressing dihydrofolate reductase activity or cos: cos ori of lambda phage: origin of replication Ap ^r : region expressing ampicillin resistance SV-dhfr: SV-40 region expressing dihydrofolate reductase activity TK: region expressing thymidine kinase activity gpt: xanthine guanine phosphoribosyl The cosmid vector pDcos Ap ^r / ori / gpt / TK, which has a cosmid vector that expresses transferase activity, has two cos, and each cosmid has a length of 1 by cutting with 2 types of restriction enzymes. A novel cosmid vector characterized in that the right arm and the left arm can be equimolar to each other. 2. The method according to claim 1, which contains a gene sequence for expressing drug resistance or a specific physiological function for determining the insertion of a gene fraction derived from a cosmid vector.
A novel cosmid vector according to the item. 3. The novel cosmid vector according to claim 2, which has a gene sequence capable of expressing ampicillin resistance, tetracycline resistance, neomycin resistance or chloramphenicol resistance as drug resistance. 4. As specific physiological functions, aminoglycoside 3'-phosphotransferase (neo) activity, thymidine kinase (TK) activity, adenine phosphoribosyl transferase (APRT) activity, hypoxanthine anine phosphoribosyl transferase. (HGPRT) activity, xanthine guanine phosphoribosyl transferase (XG
The novel cosmid vector according to claim 2, which has a gene sequence capable of expressing PRT or gpt) activity or dihydrofolate reductase (dhfr) activity. 5. The novel cosmid vector according to claim 2, which contains a gene fraction having a replication function derived from Col EI.