JP2679711B2 - Improvement of 5'upstream region of type A inclusion body (ATI) gene of box virus and improvement of foreign gene expression vector by 3'downstream region of the gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to an improved promoter corresponding to the 5 ′ upstream region of a poxvirus type A inclusion body gene, and a vector for expressing a foreign gene containing the promoter. And are useful in the production of recombinant vaccinia virus.

[Conventional technology]

Currently used vaccines are divided into live attenuated vaccines that use live attenuated viruses or bacteria and inactivated vaccines that contain inactivated viruses or bacteria.

The live attenuated vaccine induces not only a humoral immune reaction but also a cell immune reaction, and has the advantages that its efficacy is strong and the manufacturing cost is low. However, even though it is attenuated, it sometimes has the drawback of causing side effects. In addition, there are still pathogens that have not been attenuated and are not universal vaccines.

Inactivated vaccines are relatively safe, but have the drawback of being weakly effective.

In addition to these conventional vaccines, the development of vaccines that make full use of gene recombination technology is in progress. Part 1
In one method, the antigen gene of the pathogen is incorporated into a plasmid, bacteria such as Escherichia coli, yeast, etc. are transformed with this plasmid, and these transformant microorganisms are cultured to produce a large amount of the antigen protein, which is then recovered.・ Purify to produce vaccine. In this method, the vaccine can be produced if a small amount of the antigen gene is available at the beginning,
Moreover, the vaccine has the characteristic that it has no pathogenicity. On the other hand, however, there are unsolved problems in the method for purifying the antigen protein, the selection and development of the adjuvant used with the antigen protein, and the like.

In another method for producing a vaccine using genetic recombination technology, an antigen gene derived from a pathogen is incorporated into a nonessential region of a vaccinia virus gene to produce a recombinant vaccinia virus containing a foreign antigen gene. And use this virus as a live vaccine. This vaccine essentially falls into the category of conventional live vaccines and has the advantages of the above-mentioned conventional live vaccines. Furthermore, in this method, by using an attenuated virus as the vaccinia virus as a vector, a safe vaccine with low toxicity can be produced, and according to this method, a vaccine against all pathogens can be used in principle. There is an advantage over conventional live vaccines that they can be manufactured.

According to the typical procedure of this method, first, a gene encoding an antigen protein from a target pathogen (antigen gene)
Is excised and cloned in a plasmid, for example a conventional E. coli plasmid. On the other hand, a nonessential gene region of vaccinia virus or a virus closely related to it is cut out and inserted into a plasmid such as an E. coli plasmid. Then, the above-mentioned antigen gene is inserted into the non-essential gene of the plasmid containing the virus-derived non-essential gene region to prepare a recombinant plasmid. Next, a virus-derived non-essential gene region interrupted by the antigen gene in this recombinant plasmid, and a vaccinia virus containing the desired foreign antigen gene by recombination with the corresponding region of the gene of vaccinia virus as a vector. obtain. If the foreign antigen gene can be expressed in an amount sufficient to immunize a human or other animal host when the virus is inoculated, the virus can be used as a live attenuated vaccine.

By the way, virus attenuation and immunization are contradictory events, and a cooperating virus promoter is required to sufficiently express a foreign gene in an attenuated strain.

The _7.5 kd protein promoter (P _7.5 promoter) is currently known as a promoter for recombinant vaccinia virus (CeLL 25 , pp805-813 (1981) Venk).
atesan, S., BMBaroudy, and B. Moss Distinctive nucle
otide sequences adjacent to multiple initiation an
d termination sites of an early vaccinia virus gen
e). The recombinant vaccinia virus prepared by inserting a foreign antigen gene downstream of the P _7.5 promoter induces a sufficiently high antibody in small animals such as rabbits and mice, and is successful in protection against infection. However, when inoculated into chimpanzees, it is also known that the antibody did not induce a sufficient amount of antibody and protection against infection failed (Nature 328, pp721-723 (198).
7) Shiu-Lok Hu, et al. Effect of immunization with
a vaccinia-HIV env recombinant on HIV infection o
f chimpanzees).

[Problems to be solved by the invention]

The present inventors have found that poxvirus type A inclusion bodies (ATI)
The nucleotide sequence of the gene promoter (sometimes abbreviated as P _ATI ) was determined (see Japanese Patent Application No. 62-223972), and this promoter is a virus promoter that is much stronger than the P _7.5 promoter. However, as shown in FIG. 1, the P _ATI promoter contains a methionine codon ATG at its 3 ′ end, and therefore a nucleotide sequence starting from this codon and continuing downstream is expressed. As a result, when an exogenous structural gene is linked downstream of this promoter to construct an expression plasmid, the produced protein is a fusion protein of the N-terminal portion of the A-type inclusion body protein and the target protein. Therefore natural P _ATI
With the use of a promoter, the expression product must be further processed to obtain the protein of interest.
Accordingly, the present invention aims to improve the natural P _ATI promoter so as to provide an improved promoter which is more potent and can produce the desired protein in a natural form rather than as a fusion protein. And
Furthermore, it is intended to provide an improved expression vector containing the untranslated 3'region of the type A inclusion body gene together with this promoter.

[Means for solving the problem]

The present inventors have conducted research to solve the above-mentioned problems, and as a result, AT existing at the 3 ′ end of the natural P _ATI promoter has been detected.
An expression vector capable of producing the target natural protein while maintaining the activity of the natural promoter by destroying the translation initiation codon by a method such as replacing at least one of the bases in the G codon with another base is obtained. It was found that A was added downstream of this improved plasmid.
It was found that by arranging the region containing the 3'untranslated region of the type inclusion body gene, a stronger expression vector capable of expressing the foreign gene inserted between them can be obtained.

The present invention therefore comprises an improved promoter corresponding to the promoter of the poxvirus type A inclusion body gene, the methionine codon at the 3'end of which is replaced by one or more other nucleotides; and this promoter Depending on the case, it further contains a region containing the 3'untranslated region of the type A inclusion body gene downstream of this, and a foreign structural gene inserted between the promoter and the region containing the 3'untranslated region is The present invention provides an expression vector that can be expressed in.

[Specific explanation]

The poxvirus type A inclusion body gene containing the promoter region and 3'untranslated region of the poxvirus type A inclusion body gene used in the present invention has already been cloned, and Escherichia coli transformed with a plasmid containing this gene has been transformed into Escherichia coli. (Escherichia coli) JM109
-1323 has been deposited with the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology as Microindustrial Research No. 1459 (FERM BP-1459). Therefore, the promoter and the like of the present invention can be produced starting from such a plasmid.

The promoter of the present invention corresponds to the promoter of the poxvirus type A inclusion body gene, and the methionine codon at the 3'end thereof, that is, the translation initiation codon ATG is changed to one or more other nucleotides to initiate translation. Is no longer possible. As such a nucleotide, at least one of the bases ATG may be changed to another base. As an example of such a change, the case where the third base G is changed to A will be specifically described in the present invention. In addition, chloramphenicol acetyltransferase (CA) was used as an exogenous structural gene to confirm that this promoter functions.
T) Use the gene.

Next, the present invention will be described more specifically by way of examples.

In addition, the reaction conditions of various enzymes were set as follows during implementation.

EcoR I reaction solution: 100 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 50 mM NaC
l, 7mM 2-Mercaptoethanol Enzyme amount: 1 unit per 1μg of substrate Condition: 120 minutes at 37 ° C Hind III reaction solution: 10mM Tris-HCl (pH8.0), 7mM MgCl ₂ , 60mM NaCl Enzyme amount: substrate 1μg 5 units per condition: 120 minutes at 37 ℃ Klenow fragment Reaction solution: 7mM Tris-HCl (pH7.5), 20mM NaCl, 7mM MgCl ₂ ,
0.1 mM EDTA Enzyme amount: 1 unit per 1 μg of substrate Condition: 30 minutes at 37 ℃ Acc I Reaction solution: 10 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 30 mM NaC
l, 7mM 2-Mercaptoethanol Enzyme amount: 5 units per 1μg of substrate Condition: 120min at 37 ℃ Sma I reaction solution: 10mM Tris-HCl (pH8.0), 7mM MgCl ₂ , 20mM KCl,
7 mM 2-mercaptoethanol Enzyme amount: 2 units per 1 μg of substrate Condition: 120 minutes at 37 ° C Nru I Reaction solution: 10 mM Tris-HCl (pH8.0), 7 mM MgCl ₂ , 150 mM KC
l, 7mM 2-Mercaptoethanol Enzyme amount: 1 unit per μg of substrate Condition: 120 minutes at 37 ℃ Sac I reaction solution: 10mM Tris-HCl (pH7.5), 7mM MgCl ₂ , 20mM KCl,
7 mM 2-mercaptoethanol Enzyme amount: 1 unit per 1 μg of substrate Condition: 120 minutes at 37 ° C BamHI Reaction solution: 10 mM Tris-HCl (pH7.5), 7 mM MgCl ₂ , 150 mM KC
l, 7mM 2-Mercaptoethanol Enzyme amount: 5 units per 1μg of substrate Condition: 120min at 37 ℃ Exo III Nuclease reaction solution: 50mM Tris-HCl (pH7.6), 1mM MgCl ₂ , 1mM 2-mercaptoethanol enzyme Amount: 24 units per 5 μg of substrate Condition: 10 minutes at 37 ° C (reaction solution is extracted at 1 minute intervals) SI nuclease reaction solution: 30 mM sodium acetate (pH 4.6), 100 mM NaCl ₂ , 1
mM ZnSO ₄ enzyme amount: 0.1 unit for 5 μg of substrate Condition: Sma I linker ligation at 37 ° C. for 30 minutes 1 to 2 μg of blunt-ended DNA for 1 μg of phosphorylated Sma I linker was ligated with Takara Shuzo KK ligation kit React for 12 hours at 16 ℃.

Kpn I linker ligation 1 μg of phosphorylated Kpn I linker and 1 to 2 μg of blunt-ended DNA are reacted with a ligation kit manufactured by Takara Shuzo KK at 16 ° C. for 12 hours.

T4 DNA polymerase reaction solution: 70 mM Tris-HCl (pH7.4), 10 mM MgCl ₂ , 5 mM DTT Enzyme amount: 3.7 units per 10 μg of DNA (Pharmacia T4
DNA polymerase) Condition: 37 ° C for 15 minutes In addition, selection of the target plasmid after ligation of DNA fragments
Escherichia coli JM109 strain was transformed with the ligation reaction mixture, plated on LB medium, Escherichia coli was grown from the resulting colonies, and colonies were selected according to the cleavage pattern of the extracted DNA with a restriction enzyme.

Example 1. Mutation of P _ATI promoter (Fig. 2) 5 µg of plasmid pC6 (reference example) was transformed into Sac I and BamH I.
Digested with Exo III nuclease in the direction of the digestion with BamH III, and the single-stranded portion was converted to SI
The ends were blunted with 0.1 units of nuclease and Klenow fragment unit of DNA polymerase, and ligation was performed with T4 DNA ligase. Thus the plasmid p13C6-
2-32 was obtained.

About EcoR I-Taq I from this plasmid p13C6-2-32 containing the poxvirus type A inclusion body (ATI) gene
Make a 600 bp DNA fragment blunt-ended with Klenow fragment and pU
A plasmid containing the promoter region (P _ATI ) of the ATI gene by cloning into the Hinc II site of C18
We obtained p18ATIpro536. This plasmid was designated Hind III and Ec
600 bp digested with oR I and containing the P _ATI promoter
The HindIII-EcoRI DNA fragment of was isolated by agarose gel electrophoresis. On the other hand, phage M13mp18am4 (Anglian Bi
otechnology Ltd.) double stranded DNA (dsDNA) was digested with Hind III and EcoR I to linearize it, and the above-mentioned Hind III-Ec
The oR I fragment was ligated with T4 DNA ligase, circularized, infected with E. coli JM109, and white plaque formation was observed in the presence of Xgal and IPTG. The dsDNA recovered from the white plaques was identified as Hind III by the insertion of the promoter fragment.
Confirmed by double digestion of EcoRI. SsDNA (single-stranded DNA) of phage M13mp18am4-ATIpro536 containing promoter
Was prepared from the same plaque and used as a template DNA. The following experimental procedure is based on a commercially available kit (Oligonuculeotide site-dir
ected mutagenesis in M13; Anglian Biotechnology Lim
Ited) experimental manual. Primer as mutation primer ATI2 21mer 5'-AATAAATAGAGGTCACGAAC
C-3 'was used. 8th base A of primer ATI2
Is the translation initiation codon in the nucleotide sequence of ATI promoter 536
It corresponds to the position of the last base G of ATG. M13mp as a mold
A mismatch with 18 am4-ATIpro536 ssDNA will occur at this position. 10 pmole of primer whose phosphorylation of the 5'end was phosphorylated was added to 1 μg of template DNA.
The annealing reaction was started in boiling water in the presence of Cl (pH 8.0) and 10 mM MgCl ₂ , and allowed to stand until it was cooled to room temperature. Chain extension and ligation were continued. 1μ of 100mM Tris ・ Cl (pH8.0), 100mM MgC in 10μ of reaction solution
l ₂ , 1μ 5mM rATP, 1μ 5mM dNTPs, 1μ 1
After adding 00 mM DTT and 4 μl of distilled water, the mixture was placed on ice, 10 units of T4 DNA ligase and 1 unit of Klenow fragment of DNA polymerase were added, and the mixture was incubated at 12 ° C. for 16 hours. E. coli was transformed and replicated with the hybrid DNA. At this time, mismatch rep as competent cells
Air system defective strain HB2155, non-suppresso as indicator strain
Using the r strain HB2151, only the (-) chain having no amber mutation was selectively grown. The (+) chain cannot grow in the non-suppressor strain because it contains an amber mutation. When DNA was extracted from the plaques specifically selected and the nucleotide sequence was determined, a point mutation from ATG to ATA occurred in the DNA extracted from 2 plaques out of 7 plaques. Phage M13mp18 with mutation in ATI promoter
-ATIproA2-9 dsDNA, which was used for HindIII and EcoRI
A DNA fragment containing the mutated ATI promoter was obtained by digesting with.

On the other hand, the plasmid pUC18 was linearized by digesting with Hind III and EcoR I, and the above DNA fragment was added to T4 DNA.
Plasmid p18ATIpr by ligation with ligase
oA2-9 was obtained. Hind this plasmid p18ATIproA2-9
It was digested with III and EcoRI to obtain a DNA fragment containing a promoter having a mutation. On the other hand, SV40 promoter and C
Plasmid pSV ₂ CAT containing AT gene (Reference Molecular
and Cellular Biology 2 , pp1044−1051,1982; Gorman
et al. “Recombinant genomes which express chloramp
henical acctyltransferase in mammalian cells ") is digested with the restriction enzymes Acc I and Hind III to remove the Acc I-Hind III DNA fragment containing the SV promoter and enhancer, and the linearized plasmid is inserted through the Sma I linker. Circular closure gave the plasmid pSV ₀ CAT.

After digesting p18ATIpro536 with HamHI, blunt-ended with Klenow fragment and ligated with T4 DNA ligase to obtain plasmid p18ATI.
We obtained pro536ΔB. Use this plasmid for Hind III and Sm
It was digested with a I and blunt-ended with Klenow fragment to obtain a DNA fragment containing P _ATI promoter 536. Also, p18ATIproA2
After -9 was digested with Hind III and Sma I, it was blunt-ended with Klenow fragment to obtain a P _ATI promoter A2-9-containing fragment. Then linearized by digesting the plasmid pSV ₀ CAT in Sma I, P _ATI by inserting the fragment P _ATI promoter 536 containing fragment and P _ATI promoter A2-9 containing fragments using this T4 DNA ligase Downstream of the promoter
Each as an expression plasmid containing the CAT structural gene was obtained pSV ₀ -ATICAT536 and pSV ₀ -ATICAT A2-9.

The resulting plasmids pSV _0- ATICAT536, and pSV ₀
-ATICAT A2-9 was transfected into vaccinia virus infected cells and promoter activity was compared by CAT assay. The CAT assay is a method of promoter activity established by Gorman (supra) that uses chloramphenicol acetyltransferase (CATase).
The ratio of acetylation of the substrate chloramphenicol by CAT activity was used as an indicator of promoter strength. The method was as follows. pSV ₀ −ATICAT536,
pSV ₀ -ATICAT A2-9 and pUC18 plasmid DNA
Were purified as closed circular DNA by CsCl ultracentrifugation, and 0.1 μg, 0.5 μg, and 1.0 μg of pSV ₀ -ATICAT53, respectively.
6 and pSV ₀ -ATICAT A2-9 were respectively added with pUC18 as a carrier DNA at 9.9 μg, 9.5 μg, and 9.0 μg to make a total amount of 10 μg, and then aggregated and precipitated with calcium phosphate. On the other hand, CV-1 cells (cell line derived from African green monkey kidney) were used as animal cells: 8 × 10 ₅ cells were cultured overnight in a plastic culture dish with a diameter of 6 cm, and vaccinia virus WR at a multiplicity of infection (moi) of 30. The strain was infected. PBS (phosphate buffer) after 1 hour
Washed twice with MEM medium containing 5% FCS, then add 300 μl of DNA data aggregated by the calcium phosphate method, leave at room temperature for 30 minutes, and add 10 μm of MEM containing 5% FCS.
The medium was kept warm at 37 ° C. After 5 hours, change the medium once and collect the cells 24 hours after infection.
After solubilization in μ 0.25 M Tris · Cl (pH 7.8), 0.5% TritonX-100, ultrasonic treatment was performed to prepare a cell lysate. 20μ
Cell lysate, 70μ 1M Tris ・ Cl (pH7.8), 20μ
The reaction solution (150 μm) containing 4 mM acetyl CoA, and chloramphenicol labeled with 0.5 μm ¹⁴ C was incubated at 37 ° C. for 30 minutes. The reaction solution was extracted with ethyl acetate, developed by thin layer chromatography (TLC), chloramphenicol and acetylated chloramphenicol were separated, and the TLC plate was autoradiographed. Next, the portion corresponding to the spot on the TLC plate was cut out, and the amount of the label was measured by liquid scintillation. Table 1 shows the results.

From this result, the mutant promoter showed a promoter activity equal to or higher than that of the natural promoter.

The expression product of the natural promoter is N of ATI protein.
While it is a fusion protein of the end part and CAT protein,
The microorganism expressing the mutant promoter of the present invention is presumed to be a CAT protein without an ATI protein portion.

Example 2. Comparison of expression of promoters P _7.5 and P _ATI (Fig. 2) Plasmit pPro18 containing P _7.5 promoter (Reference The EMBO Journal Vol. 6 , noll, pp3379-3384 (198)
7); Effect of the recombinant vaccinia viruses tha
t express HTLV-I envelope gene on HTLV-I infecti
on) and the plasmid pSV ₀ CAT, and treating them in the same manner as described above, the expression plasmid pSV ₀ − containing the CAT structural gene downstream of the P _7.5 promoter.
I got 7.5 KCAT.

Plasmid p of the plasmid pSV ₀ -7.5KCAT and the
Using SV ₀ -ATICAT A2-9, the CAT gene was expressed by the method described above, and the expression level was measured by the CAT assay, whereby the mutant ATI promoter A2- of the present invention was expressed.
The expression ability of 9 and 7.5K promoter was compared. However, the amount of transfected DNA was 1 μg each, and 9.0 μg of pUC18 was added as a carrier DNA, and the total amount was 10 μg, and three independent experiments were performed. Table 2 shows the results.

As a result, the mutant promoter of the present invention has a CAT activity, that is, a promoter activity of 4 to 5 as compared with the 7.5K promoter.
It was shown to be 8 times higher.

Example 3 Enhancement of expression by introduction of 3'-terminal region of ATI gene (Fig. 3) Plasmid pHA13.1 (The EMBO Journal Vol. 6 , No. 6 , containing the hemagglutinin (HA) gene of vaccinia virus).
11,3379-3384,1987) was digested with the restriction enzyme Nru I to linearize it. On the other hand, the plasmid p18ATIpro A2-9 containing the mutated ATI promoter was digested with Hind III and EcoR I, and the 600 bp DNA fragment containing the promoter was Klen.
The fragment was blunt-ended with the ow fragment and ligated with the above linearized plasmid by T4 DNA ligase to obtain a plasmid pSF in which the HA gene was interrupted by a mutant promoter.

The region between the HA gene of this plasmid pSF and the downstream end of the promoter was cut with a restriction enzyme Sma I to linearize it.
On the other hand, the plasmid pSV ₂ CAT containing the CAT gene was
790 bp containing CAT gene digested with II and Sau3AI
The HindIII-Sau3AI DNA fragment was obtained. These were blunt-ended with Klenow fragment and ligated with T4 DNA ligase to obtain an expression plasmid pSFCAT containing a mutant PATI promoter and a CAT gene between the interrupted HA genes.

Next, the above-mentioned plasmid pSF was linearized by cutting with a restriction enzyme KnI. On the other hand, the plasmid p13B20 containing the ATI gene (origin: described in Reference Example) was used as a restriction enzyme EcoR V.
And 3'of ATI gene by digestion with Cla I
An EcoR V-Cla I fragment of about 1.0 kb consisting of a side coding region of about 400 bp and a 3'non-coding region of about 600 bp was obtained. This base sequence is shown in FIG. 1B. This was blunt-ended with the Klenow fragment and ligated to the linearized plasmid via the KpnI linker. Mutated P between HA genes thus interrupted
_The expression plasmids pSF.EC and pSF.ECR containing the _ATI promoter and the 3'end region of the ATI gene were obtained. Plasmid pSF / EC is followed by P _ATI promoter, 3'coding region, 3'non-coding region, pSF / ECR is followed by P _ATI promoter, 3'non-coding region, 3'side coding region, pSF / ECR is EcoR The V-Cla I fragment is inserted in the reverse direction. Each plasmid was linearized with Sma I, and then a CAT gene 790 bp DNA fragment was inserted. PSFCATEC and pSFCAT-ECR were obtained as plasmids containing the CAT gene.

Table 3 shows the results obtained by expressing these plasmids pSFCAT-EC and pSFCAT-ECR by the method described above and comparing them by the CAT assay.

As a result, the plasmid having the 3'-terminal region of the ATI gene downstream of the CAT structural gene showed about 30% higher expression power than the plasmid not having this. The reverse insertion also led to a decrease in promoter activity.

Although the present invention has been specifically described by using the CAT gene as the foreign structural gene, the improved ATI can be obtained by performing the same operation using any gene to be expressed instead of the CAT gene. An expression plasmid having a promoter and a further improved expression plasmid having the 3'terminal region of the ATI gene downstream of the structural gene can be prepared.

Specifically, the plasmid pSF can be digested with a restriction enzyme BamHI or SmaI, and an arbitrary structural gene can be introduced to prepare an expression plasmid for producing an arbitrary protein. Similarly, the plasmid pSFCAT
-EC can be digested with a restriction enzyme BamHI or SmaI and an arbitrary structural gene can be introduced to prepare an expression plasmid for the production of an arbitrary protein.

E. coli E containing the plasmid pSFEC of the present invention
scherichia coli / JM109-pSF ・ EC is Microorganisms Research Institute No. 9749 (FERM p-9749)
Was deposited as

Reference example Cloning of ATI gene Production of cowpox virus Infect 5 × 10 ⁸ Vero cells (cells derived from African green monkey kidney) with cowpox virus CPRO6 strain (obtained from Associate Professor Yasuo Ichihashi, Niigata Univ.) At moi (multiplicity of infection) of 0.2 hand,
Two days later, the virus was collected when the cell degeneration became remarkable. The virus was purified by sucrose density gradient centrifugation,
About 3 mg of virus was obtained.

Purification of cowpox virus DNA 3 mg of the obtained purified virus was treated with 1 mg / ml of protease K in the presence of 0.5% sodium lauryl sulfate (SDS) and 1 mM sodium ethylenediaminetetraacetate (EDTA) overnight.
Digested at ° C. To remove proteins, the mixture was extracted 3 times with phenol / chloroform and 3 times with ethyl ether. 1/10 volume of 3M sodium acetate and 2 volumes of isopropanol were added and stirred, and the aggregated DNA was wound up with a glass rod. DNA
Is TE [10 mM Tris-HCl (Tris-HCl) pH 8.0; 1 mM EDT
A]. The yield of DNA was 180 μg.

Construction of cowpox virus genomic DNA library 10 μg of cowpox virus DNA with Hind III or Sal I
mM Tris-HCl (pH 7.5), 60 mM NaCl, 7 mM MgCl ₂ , or 10 m
PUC18 plasmid DNA cleaved with Mind Tris-HCl (pH 7.5), 150 mM NaCl, 7 mM MaCl ₂ at 37 ° C. for 120 minutes and cleaved with Hind III or pUC13 plasmid DNA cleaved with Sal I, respectively.
Mixed with, 66mM Tris-HCl (pH7.5) , 5mM MgCl 2, 5mM
Dithiothreitol, 16 mM with T4 ligase in 1 mM ATP
The reaction was carried out for 16 hours. Escherichia coli strain JM103 or JM109 was transformed with the above recombinant plasmid to obtain a cowpox virus genomic DNA library. The recombinant plasmid contains a genomic DNA fragment of 1 kb to 25 kb.

Identification of DNA Containing A-type Inclusion Body Gene A plasmid containing cowpox virus DNA is introduced (transfected) into vaccinia virus-infected cells (CV-1 cells) to express the expression of the A-type inclusion body gene as an anti-A-type inclusion body antibody. Confirm by Western blotting using
DNA containing the type A inclusion body gene was immobilized.

First, 3 × 10 ⁵ CV-1 cells (cells derived from monkey kidney)
Infected with vaccinia virus WR strain at moi50
Time left. A plasmid containing cowpox virus DNA was extracted from the above-mentioned Escherichia coli 2 ml culture solution by the alkaline method, and 1
0 μg was transfected into vaccinia virus infected cells. After being left at room temperature for 30 minutes, 3 ml of a medium containing 5% fetal bovine serum was added, and the mixture was kept at 37 ° C. for 5 hours. The cell suspension was sonicated and electrophoresed on SDS-polyacrylamide gel, which was subjected to Western blotting using anti-A type inclusion body antibody. That is, the protein on the gel was electrotransferred to a nitrocellulose filter. Use a nitrocellulose filter with 10 mM Tris-HCl (pH 7.5), 0.15M NaCl, 5
% Anti-A inclusion body antibody after treatment with bovine serum albumin
The mixture was reacted in 10 mM Tris-HCl (pH 7.5), 0.15 NaCl for 1 hour at 37 ° C. Further, an anti-rabbit IgG antibody bound with peroxidase was reacted at 37 ° C. in the same solution as above. Finally, the expression of the A-type inclusion body gene was confirmed by staining with a 10 mM Tris-HCl (pH 7.5), 0.15 M NaCl solution containing 0.01% hydrogen peroxide and 0.5 mg / ml 4-chloronaphthol. As a result, it was revealed that the Sal I 22 kb fragment (designated as 0804) contained the type A inclusion body gene. From the plasmid containing this fragment in the library (designated p0804), Sal
This fragment was excised with I, and this fragment was further cleaved with a restriction enzyme, Kpn I, Sph I, Pst I or Sac I, and ligated to the pUC18 or pUC19 plasmid cleaved with the corresponding restriction enzyme to form a recombinant plasmid pB6. , pB20, pB23, pC3,
When pC6 and the like were obtained and the same operation was performed on these plasmids, the Kpn I-Sph I 9.1 fragment (plasmid pB
23), the Sac I-Sal I 8.9 kb fragment (plasmid pC3) contains the full-length A type inclusion body gene, and the Sac I-Sal I 6.2 kb fragment (plasmid pC6) contains a part of the A type inclusion body gene. It turned out.

Escherichia coli JM109-B23 containing the plasmid B23 was deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microindustry Research Institute No. 8971 (FERM P-8971). KOKEN Article No. 1459 (FERM
BP-1459) was transferred to an international deposit under the Budapest Treaty.

[Brief description of the drawings]

FIG. 1A shows the nucleotide sequence of the 5'end region containing the promoter of the poxvirus type A inclusion body (ATI) gene. FIG. 1B shows the nucleotide sequence of the 3'end region of the poxvirus type A inclusion body gene. 2 and 3 are production system diagrams of various plasmids of the present invention.

Claims (6)

(57) [Claims] 1. The following base sequence (I): An improved promoter corresponding to the promoter of the poxvirus type A inclusion body in which at least one base in the codon ATG of the methionine at its 3'end has been replaced by one or more other nucleotides. 2. The third base G of the methionine codon ATG.
The improved promoter of claim 1, wherein is replaced by another base. 3. The third base G of the methionine codon ATG.
The improved promoter of claim 2, wherein is replaced by A. 4. A plasmid comprising the improved promoter of any one of claims 1-3. 5. The plasmid according to claim 4, which is an expression plasmid. 6. The improved promoter according to any one of claims 1 to 3 upstream and the following nucleotide sequence (I
I): Which comprises the 3'untranslated region of the poxvirus type A inclusion body gene in the downstream thereof, and the foreign structural gene is inserted between the improved promoter and the 3'untranslated region. A plasmid capable of expressing a gene.