JPH0695937B2 - Method for producing recombinant vaccinia virus - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a recombinant vaccinia virus, more specifically, a DNA region that is not essential for the growth of vaccinia virus is previously identified, and the identified region is identified. To a recombinant site for producing a recombinant vaccinia virus.

(Prior art) In recent years, several infectious disease antigen codes have been added to vaccinia virus.
A method for using a recombinant vaccinia virus incorporating DNA as a multivalent vaccine has been proposed (for example, Science 229, 981-984.1985). When a recombinant virus is obtained by incorporating foreign DNA into vaccinia virus by such a method, the vaccinia virus genome region used for recombination must be a region not essential for the growth of vaccinia virus. However, since the procedure for identifying such a region is extremely complicated, the viral genome region currently available for such a purpose is a thymidine kinase gene, a hemagglutinin (H
A) gene, HindIII-cut F fragment, M fragment, and N fragment of vaccinia virus WR strain DNA (virus 36 (1), 23-33, 1986). However, in order to develop an effective multivalent vaccine against many infectious diseases, it is necessary to search for a non-essential DNA region on the vaccinia virus genome into which a foreign gene can be inserted in addition to the above, There has been a strong demand for the appearance of a general method for producing a recombinant vaccinia virus by utilizing an unknown non-essential DNA region by searching and identifying it and then utilizing that region as a recombination site.

(Problems to be Solved by the Invention) Therefore, as a result of intensive investigations aimed at establishing a general method for producing such recombinant vaccinia virus, the present inventors have found that the DNA encoding an enzyme whose enzyme activity can be easily detected.
Can be used as an exogenous DNA, the non-essential DNA region of the vaccinia virus genome can be easily searched and identified by a simple operation, and if that DNA region is used as a recombination site, recombinants can be easily selected. After finding out what has happened, the present invention has been completed.

(Means for Solving Problems) Thus, according to the present invention, a DN having a promoter function is used.
In order to produce a recombinant virus by incorporating A and an exogenous DNA that can be expressed under its control into a DNA region that is non-essential for the growth of vaccinia virus, a method according to the following procedures (1) to (5) is provided. .

(1) A first hybrid plasmid in which an arbitrary DNA fragment in the vaccinia virus genome is inserted, and a second hybrid plasmid in which a DNA having a promoter function and a DNA encoding an easily detectable enzyme under its control are linked. (2) A third fragment in which the DNA containing the promoter function DNA in the second hybrid plasmid and the DNA encoding the easily detectable enzyme is inserted into the vaccinia virus-derived DNA fragment of the first hybrid plasmid (3) After the third hybrid plasmid is introduced into cells infected with vaccinia virus to induce homologous recombination, a plaque assay is performed to obtain an enzyme activity based on the easily detectable enzyme. Isolating the recombinant vaccinia virus of (1) Having a promoter function by utilizing a site built connecting fragment of DNA encoding the easy enzyme detection under the domination and DNA having the DNA fragment or said promoter function from Ji near the viral genome
Producing a fourth hybrid plasmid incorporating DNA and an exogenous DNA that can be expressed under its control (5) Introducing the fourth hybrid plasmid into the first recombinant vaccinia virus-infected cell After causing homologous recombination, isolating a second recombinant vaccinia virus whose enzyme activity has disappeared by the same plaque assay as in (3). Each procedure is described below (see FIG. 1).

In the present invention, first, a first hybrid plasmid in which an arbitrary DNA fragment in the vaccinia virus genome is inserted and a second hybrid in which a DNA encoding an easily detectable enzyme under the control of DNA having a promoter function is ligated A plasmid is created.

The vaccinia virus used is not particularly limited, and specific examples thereof include WR strain, Lister strain, temperature-sensitive mutant strains of Lister strain (CNTM-I-423 etc.), New York B
Oard of Health strains are exemplified.

The first hybrid plasmid is prepared by incorporating an arbitrary DNA fragment obtained by cleaving the above vaccinia virus genome with an appropriate restriction enzyme into the plasmid according to a conventional method. Any plasmid may be used as long as it can insert the above DNA fragment, and specific examples thereof include pBR322, pBR325, pBR32.
Examples include 7, pBR 328, pUC 7, pUC 8, pUC 9, pUC 18, pUC 19 and the like.

The second hybrid plasmid is prepared by incorporating a DNA having a promoter function and a DNA encoding an enzyme whose enzyme activity can be easily detected into the plasmid according to a conventional method.

The DNA having a promoter function referred to in the present invention may be any DNA sequence that can effectively function as a promoter in a transcription system possessed by vaccinia virus, whether synthetic or natural, for example, Journal of Virolog.
y.Sept.1984, P662-669 promoter DNA sequence found in the vaccinia virus genome, specifically, the promoter of the vaccinia virus gene encoding the 7.5K polypeptide, encoding the 19K polypeptide And the promoter of the vaccinia virus gene encoding the 42K polypeptide, the promoter of the vaccinia virus gene encoding the thymidine kinase, the promoter of the vaccinia virus gene encoding the 28K polypeptide, and the like. .

Also, the enzyme activity is a DNA that encodes an enzyme whose activity can be easily detected
When integrated into a genomic region that is not essential for the growth of vaccinia virus under the control of DNA having a promoter function, the enzyme protein is produced in conjunction with the growth of the recombinant virus, and its enzymatic activity is easy. It refers to a type of DNA that can be detected, and refers to a DNA that encodes an enzyme that can be used to easily detect a recombinant virus and, in turn, to identify a genomic region that is not essential for the growth of vaccinia virus, depending on whether the enzyme activity is detected successfully. Specific examples of these enzymes include peroxidase, glucose oxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase,
β-galactosidase and the like can be mentioned, and these can sensitively detect the enzyme activity by adding a specific substrate.

Further, the plasmid used is not particularly limited, and the same one as in the case of the first hybrid plasmid can be used.

In the present invention, next, a DNA having a promoter function and a DNA encoding an enzyme are derived from the second hybrid plasmid.
To prepare a DNA fragment completely containing the DNA fragment and inserting it into the viral DNA portion of the first hybrid plasmid to give a third DNA fragment.
A hybrid plasmid of

In the present invention, the presence or absence of the formation of the first recombinant vaccinia virus is then confirmed by transferring the third hybrid plasmid into animal cells that have been previously infected with vaccinia virus.

The animal cells used here may be those capable of propagating vaccinia virus, and specific examples thereof include TK
^- 143 (derived from human osteosarcoma), FL (derived from human amniotic tumor), Hela
(Derived from human cervical cancer), KB (derived from human nasopharyngeal cancer), CJ
-1 (from monkey kidney), BSC-1 (from monkey kidney), RK13 (from rabbit kidney), L929 (from mouse connective tissue), CE (chicken embryo)
Examples thereof include cells and CEF (chicken embryo fiber Kaya cells).

The method for transferring the third hybrid plasmid into animal cells may be carried out according to a conventional method, for example, calcium phosphate method, liposome method, microinjection method and the like.

Whether or not the first recombinant vaccinia virus was constructed by the above series of operations may be selected by a conventional method. For example, when the β-galactosidase gene is used as the DNA encoding the enzyme, the recombinant vaccinia virus 5-Bromo-4-chloro-3-indolyl-β-D after plaques were observed in the plaque forming medium
Overlaying agarose medium containing galactopyranoside,
The plaques that turn blue by incubation at 37 ℃ can be selected.

Thus, when recombinant vaccinia virus is obtained by detecting enzyme activity as a means of selection, the vaccinia virus-derived DNA fragment used for the construction of the first hybrid plasmid is a non-essential DNA region for growth. It is shown that.

In the present invention, the non-essential DNA region or the DNA having the promoter function in the second hybrid plasmid and the ligation fragment of the DNA encoding the easily detectable enzyme under its control are utilized as sites for incorporating the fourth fragment.
Hybrid plasmid is prepared. This hybrid plasmid contains a DNA having a promoter function and an exogenous DNA that can be expressed under its control, and does not contain a DNA encoding the above-mentioned easily detectable enzyme or its D
The NA portion is modified so that it does not show enzymatic activity during expression.

Examples of the method for producing the fourth hybrid plasmid include the following methods.

(1) A method of inserting a DNA having a promoter function and an exogenous DNA that can be expressed under the control of the hybrid plasmid into which a fragment of all or part of the non-essential DNA region is incorporated.

In this method, the first hybrid plasmid can be used, and another hybrid plasmid prepared separately may be used as long as it has all or part of the non-essential DNA region.

(2) A method using a DNA portion encoding an enzyme that is easy to detect in the third hybrid plasmid.

In this method, in place of the DNA portion or the DNA having a promoter function and the DNA portion, exogenous
A method of inserting DNA or a DNA having a promoter function and an exogenous DNA, a method of separately inserting a DNA having a promoter function and an exogenous DNA which can be expressed under the control into the DNA part or the DNA part having a promoter function, etc. It is illustrated.

(3) A method using the insert fragment of the second hybrid plasmid.

In this case, a method of separately inserting a DNA having a promoter function and an exogenous DNA which can be expressed under the control thereof into a DNA part having a promoter function or a DNA part encoding an easily detectable enzyme is exemplified.

In the present invention, the second hybrid vaccinia virus of interest is then prepared by transferring the fourth hybrid plasmid into animal cells infected with the first recombinant vaccinia virus according to a conventional method. The conditions in this operation may be performed according to the case of producing the first recombinant vaccinia virus.

Since the obtained second recombinant vaccinia virus has lost the activity of the enzyme that can be easily detected, it is easily isolated by selecting a plaque showing no activity in the selection method of the first recombinant vaccinia virus. be able to.

(Effect of the invention) Thus, according to the present invention, it is possible to easily carry out the identification of a DNA region essential for the growth on the viral genome which is necessary when producing a recombinant vaccinia virus, and to identify the identified region. Recombinant vaccinia virus incorporating foreign DNA can be easily obtained.

Further, by using the second recombinant vaccinia virus obtained by the present invention as a parent strain and repeating a series of operations for other sites to be incorporated, a recombinant vaccinia virus incorporating a plurality of foreign DNAs can be easily prepared.

(Example) Hereinafter, the present invention will be described more specifically with reference to Examples.

Example 1 Construction of first hybrid plasmid containing vaccinia virus genome fragment (see FIG. 2) (1) Construction of plasmid (pNZ 84) containing part of Lister strain DNA Hind III C fragment 2 μg of pUC 19 (Pharmacia) Digested with Hind III, extracted with phenol / chloroform (1: 1), and pUC 19 cleaved by ethanol precipitation was recovered.
The 5'-terminal phosphate was removed by treatment with alkaline phosphatase, the DNA was extracted again with phenol / chloroform and recovered by ethanol precipitation. 0.2μ cleaved
g pUC 19 DNA and 0.1 µg of purified vaccinia virus (Lister strain) DNA were ligated with Hind III digested product to transform competent Escherichia coli JM 101 strain.
Bromo-4-chloro-3-indolyl-β-D-galactopyranoside 0.003%, isopropyl-β-D-galactopyranoside 0.03 mM, LB agar medium containing 40 μg / ml ampicillin for 15 hours at 37 ° C. Cultured. White colonies among the transformed Escherichia coli grown on agar medium were cultured in LB liquid medium containing 40 μg / ml ampicillin at 37 ° C. for 15 hours, and the method of Billundboim and Dolly [Nucleic Acid Research, 7 , 1513-, ( 1979)] and extracted the plasmid, and Hind
After digestion with III, a hybrid plasmid having a fragment of the same length as the original vaccinia virus DNA Hind III C fragment by 0.5% agarose electrophoresis was designated as pNZ51. The restriction enzyme map of the Hind III C fragment is as shown in Figure 3-a.

After digesting 10 μg of pNZ51 DNA with SalI and BamHI, 0.7% low melting point agarose gel electrophoresis (40 volt, 20 hours) gave approximately 3.8 kbp.
The SalI-BamHI fragment was isolated. Then, after confirming the DNA fragment by ethidium bromide staining, the gel was cut out, treated with phenol and then precipitated by ethanol to recover the DNA. Next, this SalI-BamHI fragment was digested with EcoRI, extracted with phenol / chloroform, and then precipitated with ethanol to form DNA.
Was recovered.

On the other hand, 0.2 μg of pUC 9 was digested with BamHI and EcoRI, extracted with phenol / chloroform, and pUC 9 cleaved by ethanol precipitation was recovered. The digested fragment with EcoRI prepared above was ligated to this cleaved pUC 9 and the resulting hybrid plasmid containing the approximately 1.78 kbp fragment was pNZ 84.
I named it. pNZ 84 corresponds to the first hybrid plasmid in the present invention.

(2) Temperature-sensitive Lister mutant (CNTM-I-423) Plasmid containing part of DNA Hind III E fragment (pNZ 60)
Preparation of 0.1 μg purified vaccinia virus (CNTM-I-423) DNA
After digestion with HindIII, a hybrid plasmid pNZ45 containing HindIIIE fragment was prepared in the same manner as in (1). The restriction enzyme map of the Hind III E fragment is as shown in Figure 3-b.

10 μg of pNZ45 was treated with Hind III and Sac I, and a DNA fragment of about 1.7 kbp was recovered by the same procedure as in (1). On the other hand, 0.2
μg of pUC 19 was digested with Hind III and Sac I, extracted with phenol / chloroform, and precipitated with ethanol to recover DNA. This cleaved pUC 19 was previously prepared with Hind III to Sac I.
0.5 μg of the fragment was ligated and the resulting hybrid plasmid was designated as pNZ60. pNZ60 corresponds to the first hybrid plasmid in the present invention.

(3) Temperature-sensitive Lister mutant (CNTM-I-423) Construction of first hybrid plasmid (pNZ 64) containing a part of DNA Hind III D fragment 0.1 μg of purified vaccinia virus (CNTM-I-423) DNA
After digesting with Hind III, follow the same procedure as in (1) to Hind I.
A hybrid plasmid pNZ44 containing the II D fragment was created. The restriction enzyme map of the Hind III D fragment is as shown in Figure 3-C.

After digesting 10 μg of pNZ44 DNA with EcoRI, a fragment of about 3.7 kbp was separated by 0.7% low melting point agarose gel electrophoresis (40 volt, 20 hours). After confirming the DNA fragment by ethidium bromide staining, the gel was cut out and treated with phenol. After that, the DNA fragment was recovered by ethanol precipitation.

On the other hand, 0.2 μg of pUC 9 was digested with EcoRI, extracted with phenol / chloroform, and then DNA was recovered by ethanol precipitation. The EcoRI digested fragment prepared above was ligated to the cleaved pUC9, and the resulting hybrid plasmid was designated as pNZ64. pNZ64 corresponds to the first hybrid plasmid in the present invention.

(3) Preparation of the first hybrid plasmid (pNZ 62) containing the temperature-sensitive Lister mutant (CNTM-I-423) DNA Hind III K fragment 0.1 μg of purified vaccinia virus (CNTM-I-423) DNA
After digestion with Hind III, follow the same procedure as in (1).
A hybrid plasmid pNZ47 containing the K fragment was created. The restriction enzyme map of the Hind III K fragment is shown in Fig. 3-d.

After treating 10 μg of pNZ47 with HindIII, a DNA fragment of about 5 kbp was recovered by the same procedure as in (1). On the other hand, about 0.2 μg
PUC 9 was digested with Hind III and EcoRI, extracted with phenol / chloroform, and ethanol precipitated to recover DNA. This cleaved pUC 9 was previously prepared with Hind III fragment 0.5
After μg was mixed and the adhesive ends were made blunt ends by DNA polymerase treatment, phenol / chloroform extraction was performed again, and DNA was recovered by ethanol precipitation. The recovered DNA was ligated and the resulting hybrid plasmid was named pNZ62. pNZ62 corresponds to the first hybrid plasmid in the present invention.

Example 2 Construction of a second hybrid plasmid (pNZ 76) in which a DNA having a promoter function and a DNA encoding an easily detectable enzyme under its control were ligated (see FIG. 4) (1) Vaccinia virus 7.5K Dalton peptide Containing the promoter of the gene encoding (pUWP-
Preparation of 1) SalI-RsaI fragment containing the promoter of DNA encoding 7.5K dalton peptide of vaccinia virus WR strain About 0.26k
bp [Cell, 125 , 805-813 (1981)] with pUC 9 SalI-SmaI
The plasmid integrated in the part was named pUWP-1.

(2) Construction of plasmid (pNZ76) in which β-galactosidase gene was ligated to 7.5K promoter (see FIG. 4) 10 μg of pMA001 [Shirakawa et al., Gene, 28 , 127-, (198
4)] was digested with BamHI, and the β-galactosidase gene (about 3.3 kbp) was recovered by the same operation as in (1) of Example 1. On the other hand, 0.3 μg of pUC 19 was digested with BamHI, extracted with phenol / chloroform, recovered by ethanol precipitation, and ligated with the β-galactosidase gene prepared above to prepare a hybrid plasmid pNZ 66.

On the other hand, 40 μg of pUWP-1 was digested with Hpa II and EcoRI and subjected to 1.5% low melting point agarose electrophoresis (70 volts, 6 hours).
A fragment of about 0.26 kbp containing the 5K promoter was isolated, and DNA was recovered by the same procedure as in (1) of Example 1. This D
The cohesive ends of the NA fragments were made blunt by DNA polymerase. 0.3 μg of pNZ 66 was digested with Hinc II, extracted with phenol / chloroform and recovered by ethanol precipitation,
The above-mentioned about 0.26 kbp 7.5K promoter gene was ligated, and the obtained hybrid plasmid was designated as pNZ76. pNZ76 corresponds to the second hybrid plasmid in the present invention.

Example 3 Construction of a third hybrid plasmid from the first hybrid plasmid and the second hybrid plasmid (1) A hybrid plasmid in which a 7.5 K promoter and a ligation fragment of β-galactosidase DNA were inserted into the KpnI site of pNZ84. Preparation of (pNZ 92, pNZ 93) (see FIG. 5) After digesting 10 μg of pNZ 76 with Hind III and Sma I, Example 1-
A fragment of about 3.6 kbp was recovered by the same operation as in (1).
On the other hand, 1 μg of pNZ84 was digested with KpnI, extracted with phenol / chloroform, and recovered by ethanol precipitation. 0.3 μg of the cleaved pNZ 84 DNA and the fragment of about 3.6 kbp (7.5
About 0.4 μg of a promoter DNA and a ligation fragment of β-galactosidase gene) were mixed, the cohesive end was made blunt with DNA polymerase, phenol / chloroform extraction was performed, and the precipitate was recovered by ethanol precipitation. The recovered DNA was ligated with ligase to transform competent Escherichia coli JM101 strain, and LB agar medium containing 40 μg / ml ampicillin at 37 ° C.
It was grown for 15 hours. From the grown Escherichia coli, the plasmid was recovered by the same procedure as in (1) of Example 1, and BamHI was used.
A hybrid plasmid containing β-galactosidase gene fragment (about 3.3 kbp) was selected by digestion with 0.5% agarose gel electrophoresis. Then, to examine the orientation of the insert, the hybrid plasmid was digested with EcoRI and the plasmid, which was separated by 0.7% agarose electrophoresis into fragments of approximately 4.3 kbp and 4 kbp, was transformed into pNZ 92, approximately 7.5 kbp and approximately 0.8 kbp fragments. The isolated plasmid was named pNZ 93.

(2) Construction of hybrid plasmids (pNZ 85, pNZ 86) in which a 7.5K promoter-ligated fragment of β-galactosidase DNA was inserted into the XhoI site of pNZ64 (see Fig. 6) After digesting 1 µg of pNZ64 with XhoI , Phenol / chloroform was extracted, and the DNA was recovered by ethanol precipitation. On the other hand, a DNA fragment in which the 7.5K promoter and the β-galactosidase gene were ligated was recovered from pNZ76 by the same procedure as in (1) above, and about 0.4 μg of the DNA fragment was cleaved into pNZ64.
Mix 0.3 μg, make blunt ends with DNA polymerase, extract with phenol / chloroform, and then precipitate with ethanol.
The DNA was recovered. The recovered DNAs were ligated with ligase, and a hybrid plasmid was prepared by the same procedure as in (1) above. The hybrid plasmid was used in Example 1.
After recovering by the same procedure as above, after digesting with BamHI, select a plasmid carrying about 3.3 kbp of β-galactosidase gene by 0.5% agarose electrophoresis and then digesting with EcoRI for about 4 kbp.
And a plasmid with a 3.8 kbp insert was pNZ 85, approximately 3.3 k
The plasmid containing the bp and 7.5 kbp inserts was named pNZ86.

(3) 7.5K promoter and β- at the BamHI site in pNZ60
Preparation of hybrid plasmids (pNZ 87, pNZ 88) in which a galactosidase DNA ligation fragment was inserted (see FIG. 7) 1 μg of pNZ60 was digested with BamHI, extracted with phenol / chloroform, and precipitated with ethanol to recover the DNA.
On the other hand, a ligated fragment of 7.5K promoter and β-galactosidase gene was recovered from pNZ76 by the same procedure as in the above (1), and about 0.4 μg of the ligated fragment was digested with BamHI to obtain pNZ60 of about 0.1.
After mixing with 3 μg, the cohesive ends were made blunt with DNA polymerase, extracted with phenol / chloroform, and then DNA was recovered by ethanol precipitation. The recovered DNAs were ligated with ligase, and a hybrid plasmid was prepared by the same procedure as in (1) above. The hybrid plasmid was recovered by the same procedure as in Example 1, and after digestion with BamHI, 0.5%.
Select a plasmid with a β-galactosidase gene of about 3.3 kbp by agarose electrophoresis, and then digest with EcoRI, and then use 0.5% agarose electrophoresis to select a plasmid with fragments of about 4.3 kbp and 4 kbp pNZ87, about 6.5 kbp and 1.8 kbp. The plasmid having the fragment of was named pNZ88.

(4) 7.5K promoter and β- at the EcoRI site in pNZ62
Preparation of hybrid plasmid (pNZ 95, pNZ 96) in which ligation fragment of galactosidase DNA was inserted (see FIG. 8) 1 μg of pNZ62 was digested with EcoRI, extracted with phenol / chloroform, and ethanol precipitated to recover the DNA.
On the other hand, a ligation fragment of the 7.5K promoter and β-galactosidase gene was recovered from pNZ76 by the same procedure as in (1) above, and about 0.4 μg of it was cleaved with the above-mentioned cleaved pNZ62 0.3 μg.
Blunt ends with DNA polymerase, extract with phenol / chloroform, and precipitate with ethanol to give DN.
A was recovered. The recovered DNA is ligated with ligase,
Hereinafter, a hybrid plasmid was prepared by the same operation as the above (1). The hybrid plasmid was recovered by the same procedure as in Example 1, digested with EcoRI and XhoI, and then subjected to 0.5% agarose electrophoresis to obtain fragments of about 1.0 kbp and 10.1 kbp, which were pNZ 95, about 4.3 kbp and about 6.8 kbp fragments. The resulting plasmid was named pNZ96.

Example 4 Preparation of First Recombinant Vaccinia Virus (1) Preparation of Recombinant Vaccinia Virus Using Lister Strain as Parent Strain vaccinia virus Lister strain was added to RK-13 cells cultured in about 80% of a 25 cm ² culture flask. 0.1 pfu / cell was inoculated. Four
After 5 minutes, the third hybrid plasmid 1 obtained in Example 3
The 0 Pg, and dissolved in sterile water 2.2 ml, gutter high et al. (Protein, nucleic acid, enzyme, 27, 340-, 1985) of making a DNA- calcium phosphate co-precipitate by the method, the 0.5ml infected RK-13 on the cell Was added dropwise. Eagle M containing 5% fetal bovine serum by leaving it in a 7% CO ₂ incubator at 37 ° C for 30 minutes.
4.5 ml of EM was added. After 3 hours, the culture medium was exchanged and
The cells were cultured in a 7% CO ₂ incubator at 37 ° C. for 3 hours, and the cultured cells were freeze-thawed 3 times.

TK cultured in 10 cm Petri dishes for selection of recombinants
^- 143 were inoculated with the above virus solution to cells, after 30 minutes, 1% agarose, 5% fetal bovine serum, 0.03% L-glutamine pressurized Ea
Glue MEM and stack at 37 ℃, 7% CO ₂ incubator for 2
After culturing for a day, the same amount of 0.5% agarose, 300 μg / ml of 5-
Bromo-4-chloro-3-indolyl-β-D-galactopyranoside-containing PBS (-) was overlaid, and 37 ° C, 7% CO ₂
The cells were cultured in an incubator for 1 day. Recombinant vaccinia virus expresses β-galactosidase and causes 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside to turn blue. Therefore, both agarose and cells around the recombinant plaque turn blue. It developed color and was easily distinguishable from non-recombinant. Recombinants generated from this blue plaque were isolated with a sterile Pasteur pipette. The obtained recombinants were named as shown in Table 1.

(1) Preparation of recombinant vaccinia virus using temperature-sensitive Lister mutant as parent strain Attenuated smallpox strain CNTM-I-423 (temperature incapable of growth in rabbit kidney cells 40.5 ° C, hatched chicken egg urine) as temperature-sensitive Lister mutant vaccinia virus Pock size on the membrane is about 0.9m
m) was used, and the experiment according to the above (1) was carried out except that the recombinant vaccinia virus selection method was changed as follows, to obtain a recombinant producing blue plaque. Each recombinant was named as shown in Table 1.

Recombinant selection method: TK ^- 143 cells cultured in a 10 cm Petri dish were inoculated with the above plaque-forming virus, and 30 minutes later, 1% agar, 5% fetal bovine serum, 0.03% L-glutamine-added Eagle MEM was added. Stacked and 37
Incubate in a 7% CO ₂ incubator for 3 days at
0.5% agarose, containing 300 μg / ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
PBS (-) was overlaid, cultured at 37 ° C in a 7% CO ₂ incubator for 2 days, and then treated in the same manner as in (1) above.

(3) Genomic DNA of the first recombinant vaccinia virus
Analysis RK in which each recombinant vaccinia virus obtained in (1) and (2) above was cultured on a 10 cm Petri dish.
-13 cells were infected with 10 pfu / cell. Below, Rice method [Journal of Birorojii, 56, 227- (198
5)] was used to isolate the viral DNA. 0.5% after digesting 2 μg of each recombinant viral DNA with BamHI or HindIII
Fragments were separated by agarose gel electrophoresis (25V, 20 hours), and D was applied to nylon membrane by Southern method [Journal of Molecular Biology, 98 , 503- (1975)].
After transferring the NA, the nylon membrane was DN at 80 ° C under reduced pressure.
A was fixed on a nylon membrane. 4x SET [0.6M NaC
l, 0.08M Tris-HCl (pH7.8), 4mM EDTA] -10 times Denhar
After treatment with dt-0.1% SDS at 68 ℃ for 2 hours, 4 times SET-10 times Denha
rdt-0.1% SDS-0.1% Na ₄ P ₂ O ₇ -50 μg / ml denatured salmon sperm DNA
And β labeled with ³² P by nick translation
-Galactosidase DNA was added and hybridized at 68 ° C for 14 hours. After washing and drying, a nylon membrane and an X-ray film were overlapped and autoradiography was performed to confirm the presence of bands, and it was confirmed that these recombinant viruses had a β-galactosidase gene at a predetermined place. From these results, promoter DN
It was shown that the hybrid DNA in which A and the β-galactosidase gene were ligated was effective in selecting a site unrelated to the growth of vaccinia and capable of inserting a foreign gene.

Example 5 A fourth hybrid plasmid (pUNZE) containing a 7.5K Dalton promoter and a hepatitis B virus surface antigen (HBsAg) gene in a part of a vaccinia virus genome fragment.
n) (see FIG. 9) 10 μg of pBRHB adr 72 [Nucleic Acid Research, 11 , 460
1 to 4610 (1983)] was digested with XhoI and BamHI, and a 1.27 kbp fragment was recovered by 0.7% low-melting-point agarose electrophoresis, extracted with phenol / chloroform, and precipitated with ethanol to give DN.
A was recovered. Both ends of the recovered DNA were made blunt with DNA polymerase, and then EcoRI linkers were ligated. This repair D
NA of 0.5 μg and pUWP-1 of Example 2 (1) 0.2 μ
g was mixed, treated with EcoRI, extracted with phenol / chloroform, recovered by ethanol precipitation and ligated with ligase to prepare a hybrid plasmid pUWPHBs-1. The orientation of the HBsAg gene with respect to the promoter was confirmed by digesting pUWPHBs-1 with Hinc II and then having a fragment of about 0.4 kbp in 0.5% agarose electrophoresis.

After digesting 10 μg of pUWPHBs-1 with HpaII, a fragment of about 1.1 kbp was recovered by 1% low melting point agarose electrophoresis, and DNA was recovered by the same procedure as above. On the other hand, 0.2 μg pN
Z62 was digested with EcoRI, extracted with phenol / chloroform, and the DNA was recovered by ethanol precipitation, mixed with the above promoter and the 1.1 kbp fragment containing the HBsAg gene, and
After blunting with a polymerase,
After extraction with chloroform, DNA was recovered by ethanol precipitation.

This recovered DNA was ligated with ligase to prepare a hybrid plasmid. After digesting the hybrid plasmid with XhoI, the one in which a fragment of about 2 kbp was detected by 0.5% agarose electrophoresis was named pUNZEn, and the one in which a fragment of about 1 kbp was detected was named pUNZEb.

Example 6 Preparation of Second Recombinant Vaccinia Virus (1) Preparation of Second Recombinant Vaccinia Virus Incorporating HBsAg Gene Using vLO 95 as a parent strain, RK13 was prepared in the same manner as in Example 1 (1).
After infecting the cells, pUNZEn prepared in Example 5 was used as a plasmid vector and transferred into the cells by the same procedure as in (4) of Example 4 to prepare a second recombinant virus. Recombinants were selected according to (1) of Example 4 except that colorless plaques were selected from a large number of blue plaques. The recombinant vaccinia virus containing the HBsAg gene obtained in this experiment was named vLO 102.

(2) Demonstration of the presence of the HBsAg gene in vLO 102 Using vLO 102, DNA was transferred to a nylon membrane by the same procedure as in (3) of Example 4, and ³² P-labeled HBsAg was nick-translated. It was carried out by hybridizing with DNA and confirming the presence of a band at a predetermined place by autoradiography.

[Brief description of drawings]

FIG. 1 shows an outline of the procedure of the present invention, FIG. 2 is a procedure for preparing a first hybrid plasmid, FIG. 3 is a restriction map of a DNA fragment derived from vaccinia virus, and FIG. 4 is a second hybrid plasmid. Procedure to do, FIGS.
Procedure for preparing the hybrid plasmid of FIG.
The procedure for preparing the hybrid plasmid of is shown below.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display area // A61K 39/285 9284-4C (C12N 15/86 C12R 1:92)

Claims (1)

[Claims] 1. A method for producing a recombinant virus by incorporating a DNA having a promoter function and an exogenous DNA which can be expressed under the control thereof into a DNA region which is not essential for the growth of vaccinia virus, to carry out according to the following procedure A method for producing a recombinant vaccinia virus, which comprises: (1) A first hybrid plasmid in which an arbitrary DNA fragment in the vaccinia virus genome is inserted, and a second hybrid plasmid in which a DNA having a promoter function and a DNA encoding an easily detectable enzyme under its control are linked. (2) A third fragment in which the DNA containing the promoter function DNA in the second hybrid plasmid and the DNA encoding the easily detectable enzyme is inserted into the vaccinia virus-derived DNA fragment of the first hybrid plasmid (3) After the third hybrid plasmid is introduced into cells infected with vaccinia virus to induce homologous recombination, a plaque assay is performed to obtain an enzyme activity based on the easily detectable enzyme. Isolating the recombinant vaccinia virus of (1) Having a promoter function by utilizing a site built connecting fragment of DNA encoding the easy enzyme detection under the domination and DNA having the DNA fragment or said promoter function from Ji near the viral genome
Producing a fourth hybrid plasmid incorporating DNA and an exogenous DNA that can be expressed under its control (5) Introducing the fourth hybrid plasmid into the first recombinant vaccinia virus-infected cell After inducing homologous recombination, isolating a second recombinant vaccinia virus from which the enzymatic activity has disappeared by the same plaque assay as in (3).