JPH03504196A - New recombinant vaccinia virus expression vector and its selection method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] New recombinant vaccinia virus expression vector and its selection method Technical field The present invention generally relates to the construction of recombinant vaccinia virus vectors. More details In particular, the present invention relates to the use of unique vaccinia virus open reading frame expression vectors. Concerning construction and its main selection method.

Background of the invention Vaccinia virus is a useful vector for gene expression in mammalian cells It is. Its advantages include maintenance of infectivity, flexible host range, and protein processing. shipping and transportation. Transcription of vaccinia virus occurs in the cytoplasm. This is done by coding enzymes and no RNA splicing occurs, so vaccines are A chinar promoter and an undisturbed open reading frame are required. In addition, Due to the large size and lack of infectivity of the Cutinia virus genome, standard inV It is not possible to construct a recombinant using itrO cloning technology.

A two-step operation has been developed to overcome these difficulties. In the first stage is a vaccine protein flanked by sequences derived from non-essential sites on the vaccinia genome. A plasmid containing a foreign gene controlled by a promoter is constructed. No. In the second step, the foreign genetic material in the plasmid vector is transferred in vivo. It is inserted into the viral genome by homologous recombination.

However, selection or isolation of recombinant viruses produced in this way is currently not possible. Known techniques such as plaque hybridization, thymidine kinase (t k) Not easily achieved by negative selection, β-galactosidase expression, etc.

Among these, the only insertional inactivation of the tk gene is a true selection step. deer However, the disadvantage of this method is that it does not meet the following requirements: Inactivation of the viral tk gene, use of specialized tk cell lines, 5-prototype Examples include the use of mutagenic selection reagents such as oxyuridine. In addition, voluntary Targeted tk mutants occur with high frequency and additional Requires a process.

Summary of the invention Therefore, the object of the present invention is to develop a new recombinant vaccine that allows the primary selection of recombinants. An object of the present invention is to provide a virus expression vector.

Furthermore, it is an object of the present invention that the genome contains mycophenolic acid (MPA) and purine A recombinant vaccine is produced on multiple cell lines when replicated in a growth medium consisting of metabolic substrates. Vaccinia provides recombinant expression vectors for wild-type vaccinia viruses. A new method for selecting virus expression vectors will become clear from the brief description.

Brief description of the drawing These and other objects, features and many attendant points will be clearly understood with reference to the accompanying drawings. For better understanding, read the detailed explanation below.

It was.

Figure 2 shows viral genome analysis from six randomly selected GPT plaques. The results are shown below. Southern plots were prepared from HindI11 digested DNA.

On the right, the fragment size of the phage random HindI [I digest is shown in kbp. ) indicated by the arrow. (A) Southern plot with (“2P) dCTP labeling It was hybridized with a gpt gene specific probe. Lanes 1-6, respectively. Lane 7 shows DNA from BSCI cells infected with virus clones Nα1-6. DNA from uninfected BSCI cells, lanes 8 and 9 are 10% of vaccinia wild type DNA. and 1100n. (B) Vaccia virus tk gene specific probe The same Southern plot is shown hybridized with /X. In lanes 8 and 9, Vaccinia virus 5.1kbp Hindll-J fragment was observed.1. J) The fragment contains the tk gene.

FIG. 3 shows a schematic architectural diagram of the vector. Vaccinia tk gene, E, coli The DNA sequences containing the gpt gene and plasmid vector are each marked with a black frame on the drawing. , indicated by a white frame and a hatched frame. The arrows are IIK (Pl, 1), 7, 5K ( P7. 5) and the direction from the tk gene promoter. The pUC sequence is Contains the ampicillin resistance gene and the origin of bacterial replication. IIK gene start code The unique cloning site downstream of the clone is shown.

Figure 4 shows the sequence of multiple cloning sites downstream of the gene initiation codon at 11. . Frameshift mutations insert an additional G residue (arrow) downstream of the ATG codon. included by entering. Restriction site E c o RI 1S a l I 5HinclI, AceL BamHI and HpaI are the only non-repetitive Be careful. Start and stop codons are shown boxed.

Figure 5 shows β-galactin in cells infected with the recombinant virus!・Sidase expression The results are shown below. Confluent CvI cells (2,5X106) were collected at a rate of 7.5 PFU/cell. After about 24 hours of incubation, the cytoplasmic extract was was prepared, and the protein content and β-galactosidase specific activity were measured. Wi The virus vF1sB is derived from the vector pTKgpt-Fls, and the virus vF1sB is derived from the vector pTKgpt-Fls. oF1sB is derived from pTKgpt-oF1s, and vtat is psc11 It was from the origin.

T7 is virus vTF7B (this virus is bacteriophage T7 RNA polymerase) and vTFga12 (1 behind the T7 promoter) acZ expressing virus), and the cells contain 7.5PF of each of the two viruses. infected with U/cells. Prior to infection, total virus titers were redetermined. β-galak Tosidase activity is the average of two independent experiments.

Detailed description of the invention The above and various other objects and advantages of the present invention are achieved by incorporating E. coli gp into its genome. From a foreign gene whose expression is desired by the t gene and one or more of its recombinant genes. The main selection method for recombinant vaccinia expression vectors that viruses inhibit purine metabolism in the presence of sufficient amounts of unphosphorylated purine substrates. When replicated in a growth medium consisting of sufficient mycophenolic acid to cause damage, achieved by a method characterized by the formation of plaques on a number of infectious cells. .

The vector of the present invention contains a promoter that provides high level expression in the genome, and Translation start and stop codons and allows expression of partial or complete foreign genes It may include multiple restriction sites in three different frames.

Unless otherwise specified, all technical and scientific terms used herein refer to the invention has the meaning commonly understood by those skilled in the art to which it pertains. of the present invention Any methods or methods similar or equivalent to those described herein may be used in carrying out and testing. Although methods and materials can be used, preferred methods and materials are described below. below All documents listed in this document apply mutatis mutandis here. Used here unless otherwise specified The techniques are standard methods well known to those skilled in the art.

Agarose was obtained from Bethesda Research Laboratories. T4 polymerase was obtained from Pharmacia.

Enzymes were used according to the supplier's instructions. MPA is obtained from Calbiochem. It was done. Xanthine and hypoxanthine were obtained from Sigma Chemicals. M.P. A is xanthine 0. IN is dissolved in NaOH and hypoxanthine is dissolved in water. These solutions were stored frozen as 10 mg/m+ stock solutions. .

Viruses and cells. Vaccinia virus (WR) was originally an American type virus. Lucher collection, replicated in He1a cells, and using standard techniques Kett et al., DNA cloning: a practical method “Vaccinia expressing foreign genes” "Construction and Characterization of Viral Recombinants", pp. 191-211, IR, L Press, Op. (Kusford, 1985). Human tk“143 cells are 10 The cells were grown in Eagle's medium with % fetal bovine serum (FBS). Cv ■ and and BSCI cells were grown in Dulbecco's modified medium (DMEM) containing 10% FBS. ) was grown in

Formation of gpt recombinant virus. Recombinant viruses are listed in the above-mentioned market etc. +5 x 106 CVI cells ( Vaccine virus at 0.2 plaque forming units (PFU) per cell (confluent monolayer) Infected Luz. Two hours after infection, 1 ml of calcium DNA precipitate (more than 5 μg Coiled plasmid DNA, 1 μg vaccinia virus DNA and 14 μg sheared (consisting of fragmented herring sperm DNA) was added to the cells. Incubate for 15 minutes at room temperature After treatment, add 9 ml of medium (DMEM, penicillin and streptomycin). 8% FBS) was added. Change the medium after 4 hours and renew the incubation. It lasted for 36 to 48 hours.

Infected cells were resuspended with the virus stock solution in 1 ml of culture medium and frozen and thawed three times. Prepared.

Selection of gpt viruses. Regarding BSCI cells for isolation of gpt recombinants Plaque assays were performed as follows: confluent B5Cl cells were grown in gpt selective medium. <DMEM, 2.5% FBS, 25 μg/ml MPA.

250 μg/ml xanthine and 15 μg/ml hypoxanthine) The cells were incubated for 14 to 24 hours.

Virus stock solution was incubated with an equal volume of trypsin (0.25 mg/ml) for 3 hours at 37°C. Digested for 0 minutes and sonicated for 20 seconds on ice. Trypsinized virus source BSCI cells were infected using dilutions (1, 0, 10' and 10-'') of the . After 1.5 hours of incubation at 37°C, cells were treated with 1% low melting point agagar. A gpt-selective medium containing loin was overlaid. 2 days incubation Afterwards, cells were stained with neutral red (Glbco). Plaques are incubated overnight. It was easy to see after the session.

DNA preparation and analysis. The recombinant plasmid was prepared using Manispring Φ Harbor La Puss, Cold Spring Barber, New York, 1982. It was constructed and isolated using standard methods. Genome analysis of recombinant viruses For this purpose, 2.5×10 B5Cl cells were infected with material from a single plaque. The cells were grown in selective medium for 24 hours. Extract total cellular DNA and extract restriction endonuclei. Digested with Ase Hind■ and electrophoresed on a 1% agarose gel using standard procedures. The results were subjected to Southern plot analysis according to the authors' work.

Construction of recombinant plasmids. pTK61-gpt: HindI[I linker Hin of E, colt gpt gene obtained from lasmid pSV2-gpt dm-Hpa I fragment. This fragment was then inserted into the unique site of pGS61. , to obtain plasmid pTK61-gpt.

p　P　　　　], :　to the IIK gene promoter of plasmid pSC42 The adjacent HindIII and 5stI sites were treated with T4 polymerase and Xhol It was converted to the Xho1 site by ligation of a linker. Plasmid pSC42 is pUc C1al-EcoRI IIK cloned into the sph 1 site of 19 Contains a promoter fragment.

pTKgpt-Fls and pTKgpt-oFls: construction of these plasmids is explained in detail in FIG.

pTKgp t-F1a and pTKgp t-F1a: Frame shift Natural mutations were performed with standard oligonucleotides directed towards mutagenesis.

A 30-mer oligonucleotide (5'-GAC CTGCAGGAATTCCATTTATAGCATAGA-3°) Another 30 mer (5°-^CCTGC) to obtain TKgp t-F1a AGGAATTCCCA-TTTATAGCATAG^-3°) was used.

Screening for mutants derived from the upstream region of the IIK gene promoter 20 Mar Brimer (5”-GCGATGCTACGCTAGTCACA-3° ) (nucleus 232-240.198B upstream of the start codon). 11 Primary structure of the promoter region and unique cloning sites in all vectors was confirmed by standard plasmid sequencing.

BamHI fragment of pMC1871 (Shabira et al., 1983, Gene 25 :7l-82) of pTKgpt-Fls and pTKgpt-oFl Cloned into the BamH1 site.

pTKgpt-FlsSpTKgpt-F1a and 1) TKgl) t-F1a Deposited with ATCC on March 18, 1988 under accession number 67.655, respectively. ．． 67.656 and 67.657. These deposits are 3 for a period of 0 years or 5 years from the last date of request for sample of the deposit, whichever is longer. maintained in a viable state and replaced where non-viable and as required by law. made available to the public without restriction.

The Commissioner of Patents and Trademarks will have access to the deposit upon request.

pTKgpt-F2sB: Smal-5a II fragment of pMc1871 was inserted into the Hinc11 site of pTKgpt-F1a.

pTKgpt-F3sB: Convert the 5alI fragment of pMc1871 into pTKgpt - Cloned into the 5alI site of F1a.

Mycophenolic acid inhibition of vaccinia virus growth.

Mycotoxin MPA inhibits the enzyme inosine monophosphate dehydrogenase to prevent the formation of xanthine monophosphate. As a result, the pudding clench This results in an intracellular decrease in the number of cells and inhibition of cell growth. Therefore, depending on the MPA of the host cell. The treatment severely inhibits virus growth. This is the increased amount of MPA in vaccinia. It was established by testing the effect of the virus on plaque formation. in medium 25 μg/ml of MPA was added to all tested cell lines (BSCI, CVI and almost completely inhibits plaque formation in human TK””143 cells). found. In B5Cl and CVI cells, after 2 days of incubation Only a few small plaques were seen on the purple-stained monolayer (first Figure A). As a result of replacing the selective medium with normal medium, the number of plaques was similar to that of the control. formation occurred (Figure 1B), indicating that the inhibition was reversible.

Expression of E. coli gpt gene and its plaque shape in the presence of MPA No impact on development. Inhibition of new synthesis of purines by MPA In the presence of substrates for purine metabolism such as santhin and hyboxanthin, the enzyme xane Encodes chin-guanine-phosphoribosyl-transferase (XGPRT) can be overcome by cells expressing the gpt gene. Wear. Blockage of purine synthesis by MPA can also be overcome by bacterial XGPRT To determine whether plasmid pTK61-gpt could be produced, we first constructed did. In this construct, the gpt gene is replaced by the vaccinia virus gene at 7.5. controlled by a promoter derived from the promoter and adjacent to the viral tk sequence I came into contact with it. The gene 7.5 was selected because it is active during early and late stages of infection. This is because the bacterial purine salvage enzyme was thought to be produced continuously. be. This plasmid was used to allow the gpt gene to recombine into the viral tk locus. into CVI cells infected with wild-type virus. Presumed recombinants are described here. BSC in the presence of MPA, xanthine and hypoxanthine as shown Detected by I cell plaque assay. Large plaques transfer the gpt gene. It is formed only when the target recombinant is used in did. One of the recombinants is a plaque purified twice under selection conditions, and a small tumor virus strain grew. Plaque assays show that this recombinant virus is different from the wild type virus. In contrast, forming plaques on BSCI cells in the presence of selective media (Figure 1C).

The worms grown from six randomly selected plaques formed during the first selection step. Figure 2 shows the genome analysis of the virus. The Hindm fragments of all six genomes are g Contains the predicted 2,0 kbp fragment that hybridizes with the pt probe. (Figure 2A). After washing out the labeled DNA, pass through the same Southern filter. It was hybridized to a chairvirus tk specific probe (Figure 2B). this tk sequences are detected as large fragments (4,7 kbp) and small fragments (1,0 kbp) , indicating the integration of the gpt gene into the viral tk locus. first selection stage All plaques selected after the step were integrated with the selection marker, allowing virus recombination to occur. No other screening procedure is required to identify the pathogen. In this way, the book The invention provides a single step procedure for primary recombinant selection.

Insertion and expression vector pTKgpt-Flg.

Construction of pTKgpt-F1a and pTKgpt-F3a.

Using the gpt gene as a selection marker, we detected the main late 1, IK polypeptide proteins. We constructed a series of plasmids that express foreign genes controlled by promoters. (Figure 3). The 5' regulatory region of the IIK gene is located immediately upstream of the ATG initiation codon. Located within a 30kbp segment. This start codon contains within it the late mRNA map. This The area was chosen as unchanged.

The presence of an EcoRI site immediately upstream of ATG allows for multiple unique cloning sites. This facilitates the insertion of polylinkers. 0.1 or 2 guanosine residues are A Three TG-compliant vectors easily insert any sequence into the correct reading frame let These vectors also include a full-frame stop codon at the end of the polylinker. give. Three vectors (pTKgp t-Fl sSpTKgp t-F 1a and pTKgpt-F3a) of the gene start codon at 11. The downstream sequence is shown in FIG.

Vector pTKgpt-oF1s (Figure 3) is for pTKgpt-Fls is a directional isomer and therefore under the IIK gene ATG as pTKgpt-Fls. They have exactly the same sequence.

Formation of recombinant vaccinia viruses expressing β-galactosidase. Vector - to ensure normal function of the construct and to easily quantify expressed proteins. (but still lack their own stop codon) so that they can coliacZ gene fragment in frame with each of the four codons. The vector was inserted into the appropriate restriction sites. pTKgpt-FlsBSpTKgp t-F2sBSpTKgpt-F3sB and pTKgpt-oFlsB Four plasmids were obtained. Using these plasmids to create virus recombinants Built. In each case, all plaques obtained under the selection conditions were found in the agar overlay. XGa1 could be converted to its blue hydrolysis product in . this is It shows the co-expression of the selectable marker gene and the symmetrical gene, and the 1acZ gene fragment is predicted. This indicates that it was within the reading frame.

To quantify the amount of β-galactosidase produced by the viral recombinant , derived from the vectors pTKgpt-Fls and pTKgpt-oFls Two gpt viruses were plaque purified three times and small strains were grown in Cv1 cells. Ta. These strains were used to develop CVI cells at a multiplicity of 7.5 PFU of each virus. infected cells.

For comparison, the same assay was performed with 1ac driven by the IIK gene promoter. A virus (vtat) based on the vector pS011 that also expressed Z (however, its β - galactosidase has a slightly different N-terminus), and phage T7 promoter Vaccinia virus T7 RAN polymer expressing 1acZ behind the This was done using The results of this analysis are shown in FIG. Vector pTKgpt-F lsSpTKgpt-ofls and pSCll contain similar amounts of β-galactosider. The 1acZ gene is relatively independent of the orientation and type of flanking sequences in the virus. Indicates that there is a relationship. The specific activity of pure β-galactosidase is 300.000 The unit is /wg. Based on this number, bacterial enzymes produced by virus-infected cells represents over 3% of total cellular protein, which is easily detected by Coomassie blue-stained gels. in a detectable amount. In fact, infection with a virus based on pTKgpt-Fls When electrophoresing proteins from isolated cells, there is a strong barrier in the 100,000 kDa region. was observed, whereas it was not observed in the wild-type virus-infected protein. Ta. The level of β-galactosidase expression using promoter 11 is shown in Figure 5. Hybrid vaccinia virus-T7 RNA under the infection conditions described It was approximately two times higher than that achieved by polymerase.

In summary, according to the invention, the gpt gene is linked to a vaccinia promoter and a foreign promoter. Plasmid vectors with unique restriction endonucleases for gene insertion introduced inside. All selection expression cassettes are homologous due to vaccinia-derived flanking sequences It is inserted as a unit into the vaccinia virus by recombination. That is, analyzed All gpt+ recombinants also contained a foreign gene inserted into a plasmid vector. Also contains. For convenience, the flanking sequences used in this study were derived from the vaccinia tk gene. It was from the past. However, following the method of the invention, tk selection is no longer required. Therefore, any non-essential site in the vaccinia genome can be used.

The promoter selected for the vector to achieve high levels of expression is the main l The lk structure was derived from protein. However, other persons skilled in the art promoters can also be used. The mechanism of late transcription is still poorly understood; Contains a non-inverted 5' poly(A) leader, but the key sequences are at the RNA start site. Contained within a relatively small region starting approximately 30 bp upstream, the conserved TAAATG sequence Contains the translation initiation codon that is part of the sequence.

For this reason, the insertion site for the foreign gene was placed downstream of the ATG. These vectors can be useful for expressing open reading frames, so multiple cloning Sites were engineered in all three frames as well as the stop codon. The efficiency of the system is As indicated by the expression of β-galactosidase, the enzyme yield is approximately It was greater than 3%. This expression level is promoted to 7, 5, which is used in floor boxes. It is higher than that obtained using Sto et al., Salmon Ce11. Blol, 7: 253g-2544,198 It exceeded even that obtained in 7 years). Vectors described here Of course, it is usually the same as that used for bacteriophage λ in E. coli. Direct cloning and expression of open reading frames in salivary mammalian cells It can also be used for

Clearly, gpt selection has not been engineered to isolate conventional recombinant vaccinia viruses. It offers numerous advantages over conventional operations. These include One-step Braak isolation, application to various cell lines, and alternative insertions in the vaccinia genome. use of entry sites, and the absence of naturally occurring selective mutants. In addition , 5-bromodeoxyuridine used for tk selection is highly mutagenic. In contrast, mycophenolic acid showed no impact in the Ames test and the related SO8 test. It is mutagenic. Absence of mutagens ensures virus stability . The gpt selection method also applies to poxviruses, herpesviruses, and adenoviruses. Other viral vectors including viruses, retroviruses, baculoviruses, etc. It can also be used for tars.

The specific examples and embodiments described herein are for illustrative purposes only, and each Certain modifications and changes may suggest to those skilled in the art and are consistent with the spirit and scope of this application and the claimed claims. Included within the scope.

FIG, IA, FIG, 18FIG, ICRG, ID Submission of translation of written amendment (Article 184-8 of the Patent Law) September 18, 1990 Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1. Display of patent application PCT/US 89100931 , name of invention New recombinant vaccinia virus expression vector and its selection method 3. Patent applicant Address: United States of America, Virginia, Springfield, Boat, Roy Yaru, Road, 5285 Name: United States of America 4. Physician (Postal code 100) 3-2-3 Marunouchi, Chiyoda-ku, Tokyo 5. Date of submission of written amendment January 1990] 66 List of attached documents (1) Request for translation of written amendment range 1. Delete.

2. Delete.

3. Delete.

4. (Correction) The purines include xanthine, hypoxanthin, and xanthine and hypoxanthine. 15. The method of claim 14, wherein the method is selected from the group consisting of a combination of poxanthines.

5. Delete.

6. (Correction) Requests that have all of the identifying characteristics with ATCC Accession Number 67.655 The plasmid according to claim 10.

7. (Correction) Requests that have all of the identifying characteristics with ATCC Accession Number 67.656 The plasmid according to claim 10.

8. (Correction) Requests that have all of the identifying characteristics with ATCC Accession Number 67.657 The plasmid according to claim 10.

9. (a) Under the transcriptional control of the first vaccinia virus promoter encodes xanthine guanine phosphoribosyltransferase (gpt) (b) a recombinant vaccinia virus genome containing a gene containing a second virus; at least one foreign DNA under the transcriptional control of a cutinia virus promoter Consisting of fragments, the gpt and a first promoter are inserted into a first non-essential region of the genome; The foreign DNA fragment and the second promoter are different from the second non-essential region. or a vaccine characterized by being inserted into a second non-essential region of the same genome. Tinia virus expression vector.

10. (a) DNA derived from the non-essential region of vaccinia virus.

(b) the first vaccinia virus promoter and the second vaccinia virus promoter under the transcriptional control; encodes gysanthinguanine phosphoribocytransferase (gpt). DNA containing the gene, and (C) A second vaccinia virus promoter and one or more foreign genes under the transcriptional control of said second promoter and with respect to said second promoter. one or more endonuclease restriction sites that can be inserted into the frame In a plasmid consisting of DNA containing (b) and (C) when the plasmid is infected with the vaccinia virus. The vaccinia virus genome containing the non-essential region when transferred into a host cell a sufficient number of nucleotides of the vaccinia virus DNA to promote recombination by A plasmid characterized in that it is inserted into and flanked by a plasmid.

11. Claim 9, wherein the second promoter is the promoter of 7.5. vector.

12. The second promoter according to claim 9, wherein the second promoter is the promoter in 11. vector.

13. A vaccinia virus in which the non-essential region encodes thymidine kinase 10. The vector according to claim 9, which is derived from.

14. In a positive selection method for recombinant vaccinia virus expression vectors, (a) Cells were treated with mycophenolic acid and a sufficient amount to inhibit plaque formation. for the enzyme xanthine guanine phosphoribosyltransferase (gpt) a sufficient amount of at least one substrate (the sufficient amount of substrate is a purine in the presence of the enzyme) Vaccinate in a cell culture medium containing (sufficient to support metabolism) infect the virus, (b) gpt, the first and second vaccinia virus promoters, and at least one endonuclease restriction site for foreign DNA insertion. DNA to be coded (this DNA is the recombinant DNA between the plasmid and the vaccinia virus) Nucleotides derived from non-essential regions of vaccinia virus in sufficient numbers to The second vaccinia virus promoter controls the expression of gpt. and said second vaccinia virus promoter is any promoter inserted into said restriction site. (c) to control the expression of foreign DNA; The dyed cells are incubated in the medium for a period sufficient to form the recombinant vaccine virus befters. A method characterized by incubating.

15. Recombination that can be used to express one or more foreign proteins In a positive selection method for vaccinia virus expression vectors, (a) Cells were treated with mycophenolic acid and a sufficient amount to inhibit plaque formation. for the enzyme xanthine guanine phosphoribosyltransferase (gpt) a sufficient amount of at least one substrate (the sufficient amount of substrate is a purine in the presence of the enzyme) Vaccinate in a cell culture medium containing (sufficient to support metabolism) infect the virus, (b) gpt, the first and second vaccinia virus promoters in the cell; DNA that encodes and also encodes one or more foreign proteins (the DNA A is a sufficient number of vaccines to perform recombination between the plasmid and the vaccinia virus. adjacent to nucleotides derived from a non-essential region of the tinia virus, and The nearvirus promoter controls the expression of gpt and the second vaccine The virus promoter controls the expression of the foreign protein, and each of the foreign proteins are encoded in different reading frames with respect to the second promoter) Introduce a plasmid containing (C) Convert the infected cells to sufficient numbers to form the recombinant vaccinia virus vectors. A method characterized by incubating in the medium for an hour.

16. Contains two or more types of DNA fragments encoding foreign proteins, and fragments are under the transcriptional control of the second promoter, and each fragment is under the transcriptional control of the second promoter. 10. The vector of claim 9, wherein the vector is inserted in different reading frames with respect to the vector.

17. Further, a virus strain is prepared from the cells and a confluent monolayer of cells is infected with the strain. and incubating the infected cells until plaques form. The medium contains a sufficient amount to inhibit plaque formation by wild-type vaccinia virus. Sufficient amount to support purine metabolism in the presence of mycophenolic acid and gpt and the monolayer of cells is incubated in the medium with at least one substrate of cells that form plaques when infected with wild-type vaccinia virus. 15. The method according to claim 14, wherein the method is selected from the group of cells.

18. Further, a virus strain is prepared from the cells and a confluent monolayer of cells is infected with the strain. and incubating the infected cells until plaques form. The medium contains a sufficient amount to inhibit plaque formation by wild-type vaccinia virus. Sufficient amount to support purine metabolism in the presence of mycophenolic acid and gpt and the monolayer of cells is incubated in the medium with at least one substrate of cells that form plaques when infected with wild-type vaccinia virus. 16. The method according to claim 15, wherein the method is selected from the group.

19. The method according to claim 14, wherein the cells are CVI cells.

20. The method according to claim 15, wherein the cells are CVI cells.

21. The method of claim 17, wherein the monolayer of cells is B5Cl cells.

22. The method of claim 18, wherein the monolayer of cells is BSCI cells.

international search report 1-. 1mfiamAalAImal+a＾to1°PCT/US8910093 1

Claims (8)

[Claims] 1. Within its genome, E. Expression by coli gpt gene and recombinant gene Consisting of a recombinant gene into which one or more desired foreign genes have been introduced, the recombinant gene Inhibition of purine metabolism in the presence of sufficient amounts of unphosphorylated purine substrate When replicated in a growth medium consisting of sufficient mycophenolic acid to An expression vector characterized by forming plaques on cell lines. 2. Non-essential in different open frames for partial or complete expression of foreign genes It further contains a strong late promoter flanked by vaccinia sequences and numerous restriction sites. The expression vector according to claim 1, comprising: 3. Recombinant vaccinia virus was tested in the presence of a sufficient amount of unphosphorylated purine substrate. growth consisting of sufficient amounts of mycophenolic acid to inhibit purine metabolism in The plaque-forming recombinants are then allowed to replicate on infectious cell lines in culture. Method for selecting a recombinant vaccinia vector characterized by isolation using conventional techniques . 4. the purine substrate consists of xanthine, hypoxanthine and combinations thereof 4. The method of claim 3, wherein: 5. Vaccinia virus promoter and restriction endonucleus for foreign gene insertion controlled by a second vaccinia virus promoter adjacent to the crease site. E. Consists of vaccinia virus DNA containing the coli gpt gene A plasmid characterized by: 6. A plasmid according to claim 5 having the identifying characteristics of ATCC 67,655. 7. A plasmid according to claim 5 having the identifying characteristics of ATCC 67,656. 8. A plasmid according to claim 1 having the identifying characteristics of ATCC 67,657.