JPH028713B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides plasmid pNR333 or
It concerns pNR334. More particularly, the invention relates to plasmid pNR333 or pNR334 as a new vector. Furthermore, the present invention provides plasmid pNR333 or
It consists of a host-vector system consisting of pNR334 and Proteus mirabilis, and is intended to provide a new technology for genetic manipulation. Genetic engineering technology is currently undergoing rapid development, and it is expected that many effective substances will be produced by genetically engineered microorganisms in the near future. Current genetic engineering technology uses a specific plasmid as a vector, inserts a gene encoding the production of an effective substance into it, creates a new plasmid, transfers this into a host, and cultivates this host. The purpose is to produce substances. However, even if a new plasmid is transferred to a host and the host is grown by culture, if the number of copies of the new plasmid per host cell is small, the amount of active substance produced according to the number of copies will be proportional to the number of copies. Therefore, the productivity of the effective substance is extremely low, making it impractical. At present, a major drawback is that the copy number of new plasmids transferred into the host is extremely low. The present inventors searched for a plasmid with a high copy number as a vector, and conducted research to find its host, and found plasmids pNR333 and plasmid pNR334, and found that Proteus mirabilis is the optimal host. That's what I learned. The present invention relates to the plasmid pNR333 or pNR334 and to a host-vector system consisting of Proteus mirabilis and the plasmid pNR333 or pNR334. Generally, the number of copies of a plasmid in a bacterial cell is 1 to 2 per bacterial chromosome, but in the case of the plasmid of the present invention, the number of copies of a plasmid in a bacterial cell is 1 to 2 per bacterial chromosome.
The number of copies in P. mirabilis is extremely large, with 172 to 625 copies for pNR333 and 114 to 357 copies for pNR334.
This is a major feature. Plasmid pNR333 with multiple copies or
pNR334 is isolated from cultured bacterial cells, then cut with restriction enzymes, and the gene fragment that encodes the information for producing the effective substance is ligated to the cut site with ligase to form a new plasmid, which is then transferred to various microorganisms and cultured. This makes it possible to produce effective substances. In Figures 1 and 2, plasmid
pNR333 and plasmid pNR334 are described. FIG. 1 shows the genetic map of plasmid pNR333, in which the cleavage sites of various DNA restriction enzymes are indicated by the following abbreviations. Pst; cleavage site for restriction enzyme Pst Sal; cleavage site for restriction enzyme Sal Bgl; cleavage site for restriction enzyme Bgl P-A (white area) is a fragment encoding the kanamycin resistance gene (Kam);
(double shaded area) is a fragment that encodes the incompatibility gene (inc), P-C (hatched area) is a fragment that encodes the replication initiation gene (ori), and P-B and P-C are Together they constitute a replicating gene (rep). Furthermore, FIG. 2 shows the genetic map of plasmid pNR334, in which Pst, Sal, and Bgl have the same meanings as above, and only the following cleavage site is newly added. EcoR: Restriction enzyme EcoR cleavage site P-A, P-B, P-C are the same as above, P-
D (the white area between the Pst cleavage site and the Pst cleavage site) is a P-D fragment (one of the Pst cleavage fragments of plasmid pNR303 described in Example 1, and the gene having the EcoR cleavage site was used as the P-D fragment) ) and has an EcoR cleavage site in the middle. Next, the physicochemical properties of plasmids pNR333 and pNR334 are shown. Plasmid pNR333: (1) Shows the genetic map in Figure 1. (2) Using λ phage DNA as a control, the molecular weight obtained by agarose electrophoresis is 4.6 Kb. (3) Using pBR322DNA as a control, the length determined by electron microscopy is 1.3 μm±0.05 μm. (4) Drug resistance is characterized by kanamycin resistance (Kan). (5) When treated with the restriction enzyme Pst, it becomes three fragments. The molecular weight of each fragment is P
-A: 1.8Kb, P-B: 1.7Kb, P-C: 1.1Kb
Fragment PA encodes a kanamycin resistance gene, and fragment P-B encodes a gene involved in plasmid replication. Copy is P-B
Start anywhere on the fragment. The P-C fragment encodes a gene involved in plasmid incompatibility, giving plasmids the property that plasmids with similar properties cannot coexist in the same cell. (6) When treated with the restriction enzyme Sal, it is cleaved at two places, producing two fragments. (7) Restriction enzyme Bgl cuts at one site. (8) Incorporation of heterologous DNA: There are six restriction enzyme cleavage sites in pNR333 that have been identified so far, and of these, the remaining four, excluding the two below, are capable of incorporating heterologous DNA. . That is, the Pst cleavage site sandwiched between the PB fragment and the P-C fragment, and the Sal cleavage site on the P-C fragment contain genes involved in plasmid replication, and therefore foreign DNA cannot be incorporated. (9) Floating density ρ: 1.706g/ml Plasmid pNR334: (1) The genetic map shown in Figure 2 is shown. (2) Using λ phage DNA as a control, the molecular weight obtained by agarose electrophoresis is 7.6 Kb. (3) Drug resistance is characterized by kanamycin resistance (Kan). (4) pNR304 (derived from copy mutant pNR300, molecular weight is
This is a plasmid in which a Pst cleavage fragment (3.0Kb) of 23.2Kb (resistant to Ap, Km, and Sm) is inserted into pNR333. This incorporated fragment is designated as the PD fragment of pNR334. There is one restriction enzyme EcoR cleavage site above this fragment. Since EcoR is currently the most commonly used restriction enzyme, pNR334 is
It can be said that the range of use as a vector for genetic manipulation has been further expanded than that of pNR333. (5) Floating density ρ = 1.708 g/ml Proteus mirabilis is preferred as the host in the present invention. Proteus mirabilis, like Escherichia coli (E. coli), lives in the human intestinal tract, and is one of the members of the normal bacterial flora that prevents the growth of pathogenic bacteria in the intestinal tract. It is also a bacterium that provides vitamins and other nutrients to humans, and is extremely safe to use. In the present invention, Proteus mirabilis Pm171 can be cited as an exemplary strain. Proteus mirabilis Pm171
is a mutant strain newly isolated by the present inventors from a laboratory-preserved strain derived from human intestinal flora, and
By using this strain, plasmids pNR333 and pNR334 of the present invention can be produced accurately,
In addition, it is easy to copy in large quantities, making it possible to create a new host-vector system by combining this strain with plasmids pNR333 or pNR334. This strain containing plasmids pNR333 and plasmids pNR334 was obtained from Proteus mirabilis at the Microtech Institute.
Pm171, deposited as FERMP-6169. The mycological properties of Proteus mirabilis Pm171 are in good agreement with the mycological properties of Proteus mirabilis described in Virghese's Manual of Determinative Bacteriology, 8th edition, pages 327-329. It is identified by its mutated properties. (lac ^- gal ^- nic trp thy ura Tc ^S ) lac ^- ; lactose non-degradable gal ^- ; galactose non-degradable nic; nicotinic acid requirement trp; tryptophan requirement thy; thymine requirement ura; uracil requirement Tc ^S ; tetracyclinc susceptibility This strain is above Because it has a genetic variation of
It is an easy-to-handle strain for various genetic experiments, and it also has the characteristic of being easily killed in the outside world and in the human body. Next, in Examples, plasmids of the present invention
An example of the production of pNR333 and pNR334 is shown, but the present invention is not limited thereto. Example 1 Production of plasmid pNR333 Human-derived, drug-resistant (at least ampicillin, chloramphenicol, kanamycin, mercury, streptomycin, sulfonamide, and tetracycline resistant) Escherichia coli was cultured, and a classroom preservation experiment was carried out by conjugative transfer. strain, Escherichia coli K-12CSH-2 with a drug-resistant plasmid (named pNR113), which was transformed into Escherichia coli K-12CSH-2RFP ^r (pNR113) (FERM
P-6170). Plasmid pNR113 belongs to incompatibility group F and contains ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), mercury (Hg), streptomycin (Sm), sulfonamide (Su), and tetracycline (Tc). ) develops resistance;
Has conjugative transferability, molecular weight is 102Kb (kilobase)
It was hot. Furthermore, plasmid pNR113 was transferred by conjugative transfer method.
Proteus mirabilis Pm171, FERM P-
Conveyed to 6169. Add this to ampicillin (Ap)
Liquid medium (L broth) containing at a concentration of 1000μg/ml
was inoculated and subcultured 5 times at 37°C.
ml was spread on Ap-containing medium to obtain highly drug-resistant plasmid pNR300.

[Table] Table 1 shows the properties of various plasmids.
Since highly drug-resistant plasmids appear from pNR113 at a frequency of approximately 1/ ^109-10 , a plasmid with properties similar to pNR300 can be easily obtained by the above method. The β-lactamase activity (penicillin-inactive enzyme) of this strain increased 10-20 times that of the original strain;
The amount of plasmid DNA is 30 to 30% of the host bacterial chromosomal DNA.
It reached 50.6% (see Figure 3). The peak with a density of 1.700 in Figure 3A indicates the chromosomal DNA of the host bacterium, and the peak with a density of 1.711 indicates the wild-type plasmid.
This shows the DNA amount of pNR113. The peak with a density of 1.711 in Figure 3B indicates the amount of DNA of the highly drug-resistant plasmid pNR300, which is significantly larger than that of wild type pNR113.
This has increased by 23 times. As a result of investigating the molecular genetic properties of this highly drug-resistant plasmid,
It was found to be a copy mutant of pNR113.
The number of copies was 23. pNR300 is an insertion sequence (IS1, insertion
Because it contains a special base sequence (also called sequence) that makes it easy to create recombinant DNA, deletion mutants occur frequently. Therefore, when a bacterium harboring pNR300 is subcultured in the presence of Ap, deletion mutants are obtained at a frequency of approximately 1/10 ^{7 -8} . Purify plasmid DNA from this cultured bacteria and reuse it
When transferred into Pm171 by the transformation method, the deletion mutant DNA is transferred into Pm171 with high frequency because it has a smaller molecular weight than the parent plasmid pNR300. Ap
A deletion mutant pNR303 was obtained by selecting transformants using a plate medium containing . pNR303 was Ap and Km resistant, had a molecular weight of 30.5 Kb, and a copy number of 36 to 52. Since the above method is often used today in the field of genetic engineering, it is easy to obtain pNR303 and the reproducibility is extremely high. Furthermore, the DNA of pNR303 was cut with the restriction enzyme Pst, ligated with ligase, and transferred into Proteus mirabilis Pm171 by the transformation method. This operation yielded a miniplasmid containing a replicated gene and a Km resistance gene, with other unnecessary genes removed, and this was named pNR333. The plasmid DNA of pNR333 reached 24.5% of the host bacterial chromosome (Fig. 3C). The number of copies is as low as 172, and as high as 625, which is significantly higher than any known plasmid to date. In addition, the amount of plasmid DNA is 70~
It turned out to be an extremely unusual plasmid with a rate of up to 80%. Mechanism of multiple copies of plasmid pNR333: Generally, plasmid replication is controlled by repressor proteins for plasmid replication. This repressor protein is a substance produced according to the genetic information encoded by the plasmid itself, and binds to the site where the plasmid starts replication, suppressing the initiation of replication. When mutations occur in this repressor protein, suppression of replication becomes uncertain, and plasmids begin to replicate abnormally. It is thought that such a plasmid based on a repressor protein mutation cannot produce multiple copies in the presence of a normal repressor protein. A normal repressor protein can be produced in plasmid pNR308 (Ap resistant, copy number 42-68), which has the same origin as plasmid pNR333.
pNR1140 (Km resistant, copy number 1-2, obtained by cloning from wild-type plasmid pNR113, molecular weight 4.6 Kb, same as pNR333) was ligated to produce a hybrid plasmid pNR308:1140. This plasmid produces both mutated and normal repressor proteins;
The copy number is 10-12, 1/5 of the copy number of pNR308.
It was ~1/6. This reduction in copy number is due to
It is presumed that this is the result of being regulated by the normal repressor protein produced by pNR1140.
It was suggested that the mutation in pNR308 is due to a mutation in the repressor protein. This suggests that plasmid pNR333, which has the same origin as plasmid pNR308, carries a mutant gene encoding a mutated repressor protein. Furthermore, the abnormally high copy number of plasmid pNR333 is thought to be due to mutations in the repressor protein as well as mutations in the replication gene site to which this protein binds. To date, several useful vector plasmids have been reported, but the copy number is at most 10 to 20, as seen in the example of Col EI, pBR322. In contrast, the plasmids of the present invention
pNR333 has a copy number of 625, and is found to be significantly more efficient than conventional plasmids. Example 2 Production of plasmid pNR334; Plasmid pNR333 obtained in Example 1 was copied in large numbers in Proteus mirabilis Pm171 and FERM P-6169, isolated by lysis, and restriction enzyme Pst was added to the obtained plasmid pNR333. The plasmid obtained in Example 1 was added to the plasmid obtained in Example 1.
Pst fragment obtained by cutting pNR303 with restriction enzyme Pst (this fragment contains restriction enzyme
There is one EcoR cleavage site. ),
Furthermore, ligase was added for ligation. The resulting ligated DNA was transformed into Proteus mirabilis Pm171 and FERM P-6169.
A strain in which the plasmid was abnormally replicated was isolated from the bacterium that was transferred to and multiplied, and a plasmid was isolated from a culture of this strain, which was named plasmid pNR334. This plasmid pNR334 has a copy number of 114 to 357
It was individual. It was confirmed that this plasmid pNR334 shows the genetic map shown in FIG. Plasmid pNR334 has one restriction enzyme EcoR cleavage site.
Since it has only one site, it can be cut with the restriction enzyme EcoR and another gene can be ligated to this site, making it extremely useful as a gene carrier DNA.

[Brief explanation of drawings]

FIG. 1 shows the genetic map of plasmid pNR333, FIG. 2 shows the genetic map of plasmid pNR334, and FIG. host bacteria into which the highly drug-resistant plasmid pNR300 was inserted.
DNA amount (B) and DNA of host bacteria into which pNR333 was inserted
FIG. 4 is a diagram showing the amount (C) of each sample subjected to CsCl equilibrium density ultracentrifugation and measured by absorbance with ultraviolet light. P-A (white area)...Fragment encoding the kanamycin resistance gene (Kan), P-B
(double shaded area)...Fragment encoding the incompatibility gene (inc), P-C (hatched area)...
Fragment encoding the replication initiation gene (ori), P-D (white area between Pst cleavage site and Pst cleavage site)...P-D fragment, Pst...
...Cleaving site of restriction enzyme Pst, Sal...restriction enzyme
Sal cleavage site, Bgl...restriction enzyme Bgl cleavage site, EcoR...restriction enzyme EcoR cleavage site.

Claims (1)

[Claims] 1. A plasmid having the genetic map shown below.
pNR333 or plasmid pNR334. 2 Plasmid pNR333 and/or plasmid
Proteus mirabilis Pm171 containing pNR334.