JP3247972B2 - A method for preparing a plasmid having both the ability to express a large amount of retrovirus gene and the processing ability after translation, and the plasmid obtained therefrom and its expression product - Google Patents

Description

The present invention provides a method for producing an expression vector capable of expressing a retroviral gene and simultaneously performing post-translational processing, and a method for producing the same. The present invention relates to a plasmid obtained by the method and its expression product. More specifically, the gag and pol genes of a retrovirus are expressed using recombinant gene technology, and the expression product itself is processed by a protease in the expression product, and three core proteins encoded by the gag gene, That is, p
17, p24 and p15, and a method for producing a plasmid which mass-produces three enzymes encoded by the pol gene, that is, a protease, a reverse transcriptase and an integrase, individually and individually as a mature protein or an active protein, and The present invention also provides a plasmid obtained therefrom and an expression product thereof. In addition, the present invention also provides a plasmid that overexpresses the nef gene and a Nef protein that is an expression product thereof. INDUSTRIAL APPLICABILITY The present invention can be used in the fields of producing pharmaceuticals and diagnostic agents by genetic engineering, and in the fields of molecular biology, virology, medicine, pharmacy, veterinary medicine, immunology, etc. as research reagents.

Conventional technology and its problems [Definition of retrovirus] Retrovirus is a generic name of viruses belonging to the family of retroviridae in terms of taxonomy, and its main main features are envelope, single-stranded RNA.
To have a type genome, and a reverse transcriptase. The virus is spherical with a diameter of 80-100 nm and its genome consists of two linear (+)-strand RNA molecules with a molecular weight of about 3 × 10 ⁶ , these two molecules being an inverted dimer.
r). In addition, the retroviridae
There are three subfamilies: Oncovirinae, Lentivirinae, and Spoomavirinae (REF
Matthews, `` Classification and Nomenclature of Vir
uses−Fourth Report of the International Committee
on Taxonomy of Viruses, pages 124-128, S. Karge
r AG 1982). Viruses belonging to the subfamily Oncovirinae are also called RNA oncoviruses and include, for example, human T
Cell leukemia virus, feline leukemia virus, murine sarcoma virus, Moloney mouse leukemia virus, bovine leukemia virus, porcine leukemia virus, avian leukemia virus,
Avian sarcoma virus, avian myeloblastosis virus, Rouss-associated virus and the like are known. Viruses belonging to the lentivirus subfamily are known as viruses that cause slow virus infection, and include, for example, human immunodeficiency virus types 1 and 2 (hereinafter referred to as "HIV-
1 "and" HIV-2 ", abbreviated as" AIDS virus "), simian immunodeficiency virus, visna virus causing sheep encephalitis, maedi virus causing sheep pulmonary fibrosis, goat arthritis encephalitis virus, horse transmission Known anemia virus, bovine lymph node lesion virus and the like (“Cu
rrent Topics in AIDS ", Volume 1, pp. 95-117, John W
iley & Sons 1987; Advances in Virus Researc
h, Vol. 34, pages 189-215, 1988). Viruses belonging to the subfamily Spoomaviridae are also called formiiviruses, which infect mammals such as humans, monkeys, cattle, cats, etc., and formmyviruses isolated from these hosts, respectively.
Syncytial virus and the like are known. The retrovirus according to the present invention means all known and unknown viruses belonging to the retroviridae.

[Current status of basic research on retroviral genes] Retroviruses are not only important viruses from the viewpoint of malignant or fatal infectious diseases of humans and various animals, and zoonotic diseases, but also include elucidation of tumors and genetic engineering. It is a useful virus as research material. Therefore, since a large number of reports have already been made on the virus, here, for the sake of convenience, the current status of a typical retrovirus is described below: As is well known, the retrovirus is 1980.
A year ago, it was noted and studied as a material for elucidating the mechanism of carcinogenesis and from the perspective of investigating strange slow virus infections that cause intractable diseases. Since the discovery of AIDS in the United States in 1981, comparative studies between various retroviruses have been used as research materials or experimental models to establish treatment and prevention at a global level. Epidemiology, immunology, virology, molecular biology The virus has been intensively promoted, and many useful reports on the virus have already been accumulated (Annu
al Review of Immunology, Volume 6, 139-159, 1988; Mi
crobial Pathogenesis, Vol. 5, 149-157, 1988; "Vi
rology ", BNFields, etc., Volume 2, pp. 1437-1589,
Raven Press [USA] 1990). Among these, as a representative example, an outline related to the HIV-1 gene will be described below. The HIV-1 genome forms a complex with reverse transcriptase and a structural protein inside the virus particle (core), and is internalized in the virus particle together with the primer tRNA; the virus genome is composed of about 10 types of genes, Basically, three major genes encoding virion components essential for virus propagation, namely gag (group-spec), which encodes precursors of three structural proteins of the viral core.
env (envelop) gene, a pol (polymerase) gene encoding three types of enzyme precursors, and an env (envelop) encoding two types of envelope glycoprotein precursors
e) Consist of genes; these genes are arranged in order from the 5 'end to gag, pol, env, 3'end; characteristics of the lentivirus include, for example, gag in the case of the HIV genome. Pol · sor… env · nef The sequence is adjacent in the order of env · nef, and a part of the 5 ′ terminal region of the pol gene is about 5% of the 3 ′ terminal region of the gag gene in a different codon reading frame. It is thought that this overlaps with 240 bases, a frame shift occurs at the time of translation at the overlapping portion, and the stop codon is replaced to proceed with translation; and the gag gene of about 1.5 kb including the overlapping portion is included. Molecular weight from all regions
After the 55 kd precursor protein is translated, it is thought to be cleaved by proteases, i.e., processed, to become three gag proteins that function as matrix proteins, capsids and nucleocapsids, respectively, in the above order, p17, p24 and p15, respectively. ing. In addition, the expression of the entire pol gene region of about 3 kb in total length allows the above enzyme precursor (molecular weight 100 kd)
But after being produced in the form of representable fusion protein as the NH ₂ - protease, reverse transcriptase, integrase-COOH, such fusion proteins themselves, existing proteases or the same molecule derived from said viral Is cleaved or cleaved under the action of the protease activity of, so-called processing results in individual mature proteins (active proteins),
That is, protease (p12), reverse transcriptase (p66 and p51)
And integrase (p32) (Journal of Virology, Vol. 62, No. 5, 1808).
-1809, 1988). In addition, each of the above three enzymes plays an important role in the process of virus growth and maturation or in the process of provirus formation, and the following is confirmed or estimated for each of these functions: Proteases are involved in post-translational processing and the core formation or maturation process of virions, and protease activity is highly specific for the virus from which it is derived; reverse transcriptase is a fundamental step in viral growth RNA catalyzes the reverse transcription process from viral genomic RNA to DNA
In addition to functioning as a dependent DNA polymerase, it must also have a ribonuclease H activity that specifically degrades only RNA that is complementary to the DNA, and a DNA-dependent DNA synthesis activity that generates double-stranded DNA. Is known,
It is also useful and commonly used as a material for genetic recombination, for example, an enzyme for complementary DNA synthesis;
An endonuclease that acts on DNA strands and catalyzes recognition and cleavage of the integration site into the host chromosomal DNA of circular viral double-stranded DNA generated from the viral genomic RNA through the above reverse transcription process to become a provirus ("HIV and Other Highly Pathogeni
c Viruses ", pages 33-41, Academic Press, Inc. 1988.
Published in the year: “The Control of Human Retrovirus Gene Exp
ression ", pages 79-89, Cold SpringHarbor Laborato
ry Published in 1988; Cell Engineering, Volume 7 (Suppl.1), S5-S15
Page, 1988; AIDS Journal, Volume 1, Issue 3,
291-300, 1988; Aids, Volume 2 (suppl. 1), S29
-40, 1988; Chemistry and Biology, Vol. 27, No. 4, 21
8-227, 1989).

In addition, the nef gene region having a total length of about 0.4 kb has a molecular weight of about 27 kd.
Encodes a negative regulatory factor, which is thought to be involved in latent infection by inhibiting HIV genome expression and stopping HIV growth (“Virology”, BNFields et al., Vol. 2, pp. 1535-1535, Raven Press [USA], 1990).

[Current status and issues of mass production of retroviral gene products by genetic engineering] Mass production of various retroviral gene products (hereinafter referred to as "antigens") by genetic engineering is for the purpose of developing vaccines and diagnostic agents for humans and animals. And is widely practiced around the world. As a representative example, mass production of the AIDS virus antigen will be outlined below. The main antigen being studied for mass production is the precursor glycoprotein gp1 of the env gene.
60 and its processing products, such as gp120 and gp41 (“The Forefront of AIDS Control,” edited by Toshiaki Komatsu, II, 47
7-495, CMC 1989), for example, gp
Production of Baculovirus vector and insect cells (Proceedings of National Academy of Sciences [US
A], vol. 84, pp. 6924-6928, 1987), recombinant vaccinia virus and BSC-40 or HeLa cells (Nature, vol. 320, pp. 5).
37-540, 1986; ibid., Vol. 330, pp. 259-262, 1987), adenovirus vector and A549 cells ("Vaccine 89", pp. 2).
07-217, Cold Spring Harbor Laboratory 1989)
Use has been reported. Further, expression of the gp120 gene region in a CHO cell line and a plasmid in which the gp120 gene region is inserted and linked (Science, vol. 233, pp. 209-212, 1986), and production of gp120 in E. coli (Science, vol. 234, pp. 1392-1395,198
6) have been reported. Furthermore, the outline of the main known technologies relating to this is categorized and listed (hereinafter referred to as “EP” for European published patent number, “DE” for West German published patent number, “US” for US patent application number, PCT international patent publication number). Are abbreviated as “WO” and French published patent numbers are abbreviated as “FR,” respectively: (1) Techniques intended for high-level expression of genes such as gag, pol, and env (hereinafter, described according to the host used): E. coli ( EP 331961, EP 322394, DE 3727137, DE 372401
6, EP 293792, US 88-168486, US 88-218304, US 87-110
348, EP 255190, WO 87/07296, DE 3711016, EP 227169, EP
Yeast (ED 3804891, EP 322394, US 88-168486); (2) Technique for producing recombinant vaccinia virus into which genes such as gag, pol, and env are inserted: JP-A-1-148183, FR 2620030, FR 2607518, FR 26000
79, FR 2587720, FR 2596771, EP 256677, WO 87/06260, WO
87/06262; (3) technology for expressing the env gene with a viral vector: use of polyoma virus (JP-A-1-39991), baculovirus (EP 272858, EP 265785), and the like; and (4) AIDS virus antigen B Techniques for expressing the protein as a fusion protein with hepatitis virus antigen (EP 278940) are known.

By the way, apart from the technology for producing a recombinant virus that can be used as an active ingredient such as a live vaccine, in general, in industrial production of a useful protein using an expression vector, in order to ensure low production cost and high quality, large amounts of gene expression and gene expression are required. The three elements of secretion of the product outside the host and post-translational processing are extremely important, and these three elements need to be taken into account in the construction of an expression vector. In this regard, as is apparent from the above-mentioned outline of the prior art, various studies have been made on high expression, and development of a secretory production system (Journal of the Japanese Society of Agricultural Chemistry, Vol. 60, No. 5, pp. 1035-1063, 1990) )
Has also been widely promoted. However, attention has not been paid to the ingenuity of post-translational processing, although it is important to save labor in the purification step of gene products and to increase the purity of such products. Therefore, it is clear that the development of an expression system that enables post-translational processing has valuable significance. In addition, Nef protein is useful in the fields of AIDS diagnosis, elucidation of onset mechanism, treatment, etc. from the viewpoint of the antigenicity and function, and thus the establishment of mass production is expected.

Means for Solving the Problems The present inventors have conducted intensive studies in order to overcome the above-mentioned problems of the prior art, and as a result, the retrovirus gag and p
High expression of 6 kinds of proteins encoded by ol gene,
Moreover, we succeeded in processing. In other words, the present invention relates to three viral core proteins, p17, p24, and p15, which are retroviral gag gene products, and three enzymes, proteases, reverse transcriptases and integrases, which are pol gene products. The present invention has been completed by achieving the production of plasmids which can be mass-produced at low cost with a stable and high production yield as mature proteins or active proteins, individually and individually. Such achievement is achieved by making full use of gene recombination technology to match the translation frame within a gene or a direct expression vector capable of inducing a gene cDNA fragment of the retrovirus prepared so as to essentially contain a protease gene of the virus. This resulted in the production of a ligated plasmid, and the plasmid expressed a large amount of the gene product as expected, and the processing of the gene product itself by the expressed protease was confirmed. It should be noted that the direct expression means that the retrovirus gene is not indirectly expressed as a fusion protein with a heterologous protein derived from the gene previously integrated into the vector. Furthermore, the present inventors obtained a transformant by inserting the plasmid constructed by inserting and linking the cDNA of the gene, and applying the two-step culture method described below to the culture of the transformant to obtain the transformant. In culture, the cDNA
Have been found to be produced in extremely large amounts and stably, not as fusion proteins, but as individual mature proteins having specific activity, respectively. Furthermore, it has been found that such processing is due to a specific activity of a protease occupying a part of a fusion protein which is an expression product of the above gene. That is, it has been found that the processing is a phenomenon peculiar to the retrovirus protease gene. Further, the processed proteins are easy to produce and purify in large quantities,
In addition, they have found that they have excellent purity and homogeneity, and particularly have enzyme activity and antigenicity, which are extremely high in substrate specificity unique to retroviruses. In addition, mass production of Nef protein having extremely specific antigenicity was achieved. The present invention has been completed based on these findings.

According to the present invention, gag, pol, and nef genes related to retroviruses encode a gene, and a method for producing a plasmid capable of large-scale expression or mass production and processing of various proteins, and a plasmid obtained therefrom and an expression product thereof Provided. Such plasmids and the expression products of the core proteins p17, p24 and p24, as well as proteases, enzymes of reverse transcriptase and integrase, and Nef protein, as materials for the study of prevention and treatment of retrovirus infection,
For example, it is extremely useful as a reagent in the fields of genetic engineering, protein engineering, molecular biology, development of pharmacotherapy and antiviral agents for retroviral infection, and as an antigen for vaccines, diagnostics, and antibody production. It is.

The constitution of the present invention is as follows: (I) Selection of retrovirus gene and preparation of cDNA fragment thereof: With regard to the retrovirus gene, gag, pol, Genes such as retrotransposons can be used. More specifically, in the case of HIV, for example, each gene in the gag region encoding core protein, p17, p24 and p15, each gene in the pol region encoding protease, reverse transcriptase, and integrase, and the like are used. Expression of these genes requires the use of a protease gene, and at least or the shortest of the above-described genes relating to the retrovirus, at least the shortest, a retrovirus gene cDNA fragment prepared to contain the protease gene region, is expressed in large amounts. It is used by inserting and linking the reading frames in the gene so as to match. In addition, recombinant DN
In gene expression by A technology, the retroviral genome
Since it is NA, each of the above genes is used after being converted into a cDNA fragment complementary thereto. Such a cDNA fragment can be prepared from a provirus genome integrated into the host chromosome or a cloned non-integrated circular DNA. In addition, using genomic RNA extracted from virus particles as a template, it was prepared by a known conventional method using reverse transcriptase.
It is also possible to select and prepare from a cDNA library. However, in the above-described preparation, since a high-risk retrovirus is directly handled, from the viewpoint of prevention of biological disasters,
Not always easy. Therefore, in order to avoid biohazards caused by the virus and save labor in the preparation step, it is recommended to use a known and ready-made retrovirus genomic cDNA clone. That is, as seen in the review cited above, cloning of various retrovirus genomes, creation of restriction enzyme maps, determination of nucleotide sequences, and the like have already been reported by researchers all over the world. Utilization of the results is safe, simple, and desirable. For example,
Plasmid pSRA2 (Journal of Virology, 36, 50-61, 1980), a ready-made avian sarcoma virus genomic clone, plasmids pNL3-1, pNL3-2, and pNL4-3, HIV-1 proviral genomic clones (Jour
nal of Virology, Vol. 59, pp. 284-291, 1986), H.
E. coli UT481 / pNLH40, a pol gene clone of IV-1
Plasmid pNLH402 of E.coli
JM109 / pCV91 (Microtechnical Laboratories No. 3195), E.coli JM109 / p
NLH122 (Microtechnical Laboratory No. 3196) and the like are available and can be used. The cDNA fragment can be prepared by a conventional method known in the art, for example, after excising DNA of a necessary region from the above-mentioned clone with a restriction enzyme, and then purifying by phenol extraction, chloroform treatment, ethanol precipitation and the like. Restriction enzymes used for excision of DNA fragments can be appropriately selected with reference to a restriction enzyme map of each clone. For example, the above pNLH402
If it is desired to cut out a DNA fragment of the entire region of the pol gene, the restriction enzyme Hind III (Journal of Virology, Vol. 59, 284-
291 p. 1986).

(II) Construction of a retrovirus gene expression plasmid and preparation of a transformant transfected with the plasmid: The retrovirus genomic cDNA fragment prepared as described above was ligated into a gene expressed in a large amount using a conventional method, for example, T4 DNA ligase. Alternatively, a retroviral gene expression plasmid is constructed by inserting and linking into a gene direct expression vector. The term “plasmid” according to the present invention is used for convenience notation, and means a replicon that expresses a retroviral gene substantially and widely. Therefore, to construct such a plasmid, a known or commercially available expression vector, for example, a plasmid vector pSN508 series of Enterobacteriaceae (US Pat. No. 4,703,005), a yeast plasmid vector pJM105 (Japanese Patent Application Laid-Open No. 62-286930) ) And pBH103 series (JP-A-63-22098), attenuated varicella virus vector (JP-A-53-41202), attenuated Marek's disease virus vector (European Patent No. 334530), and a plasmid vector pUR290 series of E. coli (EMBO) Journal, Volume 2, 1791-17
94, 1983), pSN5182 (Journal of Bacteriology, Chapter
157, 909-917, 1984) and pT7-7 (Procee
dings of the National Academy of Sciences [USA],
82, pp. 1074-1078, 1985).
What is important in the construction of an expression vector is to link the above-mentioned gene with a gene having a high expression level. For example, when the above-described pUR290 is used, the gene is downstream of the lacZ gene, downstream of the pstS gene in pSN5182, and downstream of the oligopeptide gene derived from pT7-7 under the control of the T7 promoter in pT7-7, respectively. Is preferably linked. It should be noted that such gene insertion should be coordinated so that codon reading frames of the genes coincide with each other so that translation proceeds smoothly. For example, HIV-
1. When cDNA fragments such as HIV-2, simian immunodeficiency virus, and Moloney murine leukemia virus are inserted and linked, since the protease of such virus is encoded in the pol gene region, the pol gene has a large amount of open reading frame. Cooperate to match that of the expressed gene. In addition, the protease of avian sarcoma virus is encoded in the gag gene region, which has a different reading frame from the pol gene region, and the protease genes of human T-cell leukemia virus, bovine leukemia virus, etc., have different reading frames from the gag gene and the pol gene. . In such a case, it is necessary to devise a method for matching the mutual reading frames among the large-expression gene, the protease gene and the pol gene. By such a measure, the expression levels of various enzyme genes are guaranteed. The agreement of the reading frames can be achieved by a known method using enzymes such as a restriction enzyme, nuclease Bal31, and mung bean nuclease. In addition, the optimal recipient cells used for the purpose of obtaining a transformant by transferring the constructed expression vector are selected from susceptible host cells that permit the replication and expression of the expression vector, and at the same time, among such host cells, Cells that can easily and reliably detect the transfer of the constructed expression vector are carefully selected and used. For example, when the above-described pSN series is used as an expression vector, it is preferable to use, as a recipient bacterium, an Escherichia coli C75 strain, which can be used to select a transformant obtained by transferring the vector as a marker for drug resistance, No. 10191). When the pUR290 series and pT7-7 are used, a transformant into which this vector has been transferred can be selected using ampicillin resistance as a marker for the E. coli UT481 strain (Journal of Bacteriology,
163, pp. 376-384 (1985) and E. coli BL21 (D
E3) strain (Journal of Molecular Biology, Vol. 189, No. 1, pp. 113-130, 1986) can be used, respectively. The transfer of the expression vector into such a recipient cell can be performed by a known method, for example, the calcium chloride method (Journal of Molecule).
ar Biology, Vol. 53, pp. 154-162, 1970). In addition, the transformant into which the gag or pol gene expression plasmid has been transferred is selected from colonies positive for the marker. Next, after extracting the expression vector DNA from the selected transformant colonies, the expression vector DNA was cut with a restriction enzyme, and subjected to agarose gel electrophoresis to measure the size of the inserted and linked DNA fragment. A colony in which the presence of the fragment has been confirmed is adopted as a transformant clone expressing the retrovirus gene.
For example, when the entire pol gene derived from pNLH402 is linked to the expression vector pUR290 described above, about 4 kb
An EcoRI fragment is detected.

(III) Confirmation of retrovirus gene expression by transformant clone and mass production of various proteins by culturing the transformant: Confirmation of gene expression by transformant clone is carried out, for example, by Western blotting. The analysis can be performed by analyzing a crude extract of the clone culture. The crude extract is obtained, for example, by culturing the transformant in a known medium, collecting cells by low-speed centrifugation, treating the cells with sodium dodecyl sulfate and 2-mercaptoethanol, and then subjecting the cells to high-speed centrifugation. Can be prepared. In addition, Western blotting can be performed using a wide variety of commercially available materials in accordance with a conventional method in the following order: the crude extract is subjected to polyacrylamide gel electrophoresis; Transfer onto a nitrocellulose membrane using a blot cell; immerse the membrane in gelatin solution and block. And below
For example, if the analyte on the membrane is an HIV pol gene product, it is subjected to a primary reaction with an HIV carrier serum; after washing, it is further reacted with a peroxidase-labeled anti-human IgG antibody; After washing, color was developed with a hydrogen peroxide solution and a color former, and the band specifically reacting with the HIV carrier serum was detected.
Check gene expression. When the subject is a gene product derived from a retrovirus other than HIV, HI
The V-carrier serum is not used, and the primary reaction uses the relevant retroviral antiserum, and the secondary reaction uses an antibody against human or animal IgG derived from the antiserum. gag
Mass production of various core proteins, proteases, reverse transcriptases, and integrase enzymes by culturing transformants in which the expression of the pol gene has been confirmed is performed as follows: For preparation, for example, when the transformant is Escherichia coli, the cell concentration is 2 × 10 5 in LB medium.
Until reaching ⁹ to 8 × 10 ⁹ cells / ml, and cultured 12 to 35 hours at 30 to 40 ° C.; Next, to the culture medium 1000 previously prepared, after inoculation mixing such seed 1-10, Two-stage culture consisting of pre-culture and post-culture is performed. The preculture is performed for the purpose of growing seed cells and amplifying the expression vector, and is performed at 10 to 40 ° C for 1 to 24 hours, preferably, 15 to 37 ° C.
For 2 to 12 hours, for example, in the case of Escherichia coli, the pre-culture is terminated based on the cell concentration under culture, that is, the culture solution turbidity OD600nm = 0.1 to 2.0. Next, upon completion of such pre-culture, the culture system is transferred to post-culture. Post-culture
The transcription and translation of the enzyme gene linked to the expression vector, and the translated gene product are remodeled to ensure the processing of individual active mature proteins, while the translated enzyme gene product is transferred to the host cell. It should be set with extreme care and ingenuity to avoid random degradation and inactivation by the proteolytic enzymes of origin. The post-culture is preferably performed at a relatively low temperature as compared with the pre-culture, and at 10 to 40 ° C for 1 to 40 hours, preferably 15 to 37 hours.
C. for 3 to 35 hours. In addition, in consideration of the properties of the expression vector to be used, for example, at the start of post-culture, the promotion or induction of expression is performed by starvating the phosphate in the medium, adding and mixing an inducer in the medium, and the like. be able to. In addition, by performing the two-step culture described above, each core protein of the retrovirus, protease, reverse transcriptase, and each enzyme of the integrase are not in the form of a fusion protein or a bacterium, but independently active proteins, that is, As a separate and separate mature protein, it is usually produced in high yields of about 1-30 mg per medium.

(IV) Purification of each core protein, protease, reverse transcriptase, and integrase enzyme mass-produced by the expression vector: This step can be achieved by using a combination of known conventional methods. For example, collection of a transformant culture by a precipitant, centrifugation, filtration, etc .;
Preparation of crude extract by disruption or disruption of transformant cells using a homogenizer, etc .; purification by adsorption / desorption treatment with silica or activated carbon, salting out, precipitation with an organic solvent, etc .; ultracentrifugation, column chromatography, electrophoresis, etc. Highly purified by fractionation used; or by adsorbing and desorbing to silicic acid and activated carbon, and then purifying the gene product to be fractionated by density gradient centrifugation (JP-A-63-297) by a conventional method such as Enzyme purification is possible.

The expression vector obtained by the method of the present invention is provided in a sealed state in a small container such as an ampoule or a vial, or in a state transferred to a host. In addition, each core protein, protease, reverse transcriptase and integrase each enzyme mass-produced by the expression vector according to the present invention is in the form of a liquid, a dry powder, or adsorbed on filter paper or a membrane, such as an ampoule or a vial. Can be provided sealed in a small container. In the case of a liquid, use the necessary amount as it is, and in the case of drying, dissolve it in distilled water and return to the volume before drying, then use the required amount. In the case of adsorption on filter paper, membrane, etc., use it after wetting it with the solution specified in the instruction manual.

(V) Construction of nef gene expression plasmid, mass production / confirmation / purification / use of Nef protein: These are described in (I) to (IV) above.
Can be performed in the same manner as described above. However, since the nef gene is located at a position distant from the gag and pol genes in the HIV genome and does not require post-translational processing for the production of the Nef protein, the protein was produced as a single protein. It is more rational and labor-saving. Therefore, mass production of Nef protein can be achieved by expressing a large amount of the nef gene alone without considering processing. That is, the nef gene expression plasmid is prepared by inserting and linking the nef gene cDNA fragment into a large expression gene or a direct gene expression vector. The nef gene cDNA fragment can be obtained from the plasmid pNL4-3 (Journal of Virgin) containing the nef gene region in the HIV genome or the nef gene cDNA.
ology, Vol. 59, pp. 284-291 (1986)) and psmid pN
It can be prepared using LH152 (Microtechnical Laboratory No.).

Hereinafter, specific examples of the present invention will be described with reference to examples. However, the present invention is not limited only to the following examples.

Reference Example 1 Measurement of reverse transcriptase activity: The composition of the reaction solution was 50 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, 10 mM magnesium chloride, 3 mM dithiothreitol, 0.1% (W / V). Nonidet P-40 (manufactured by Shell Sekiyu Kabushiki Kaisha [USA]) 20 μg / ml (rA) _n · (dT) _12-18 (manufactured by Pharmacia Kabushiki Kaisha), 0.5 mM TTP (thymidine triphosphate),
50 μl including 1 μCi of [ ³ H] · TTP including 5 μl of the sample was incubated at 37 ° C. for 10 minutes. The reaction solution was immediately cooled on ice, filtered through filter paper DE81 (manufactured by Whatman [UK]), and the filter was washed well with 5% sodium phosphate solution, further washed with water, washed with ethanol, and dried to obtain liquid scintillation. The cpm was measured with a counter.

Example 1 Construction of an Expression Vector Linked with Lentiviral pol Gene Insertion: Plasmid pNL4-3 Having HIV Provirus Genomic DNA (Journal of Virology, 59 (2): 284-
5 μg, 5 μl of Hind III, 20 μl of 5
× RM (50 mM Tris-HCl, pH 7.5, 35 mM MgCl ₂ , 300 mM NaC
l), make up the total volume to 100 μl with distilled water, and add 1
Time, after incubation, TE (10 mM Tris-HCl, pH 7.5,
Extraction was performed with phenol saturated with 1 mM EDTA, and the aqueous layer was treated with chloroform and precipitated with ethanol. 10μ precipitate
1 μl of a solution dissolved in 1 l of TE, and a plasmid cleaved with Hind III and treated with alkaline phosphatase, pHSG398
0.1 μg (1 μl) of DNA and 2 μl of 10 × ligation buffer (660 mM Tris-HCl, pH 7.6, 66 mM MgCl ₂ , 100 mM
M DTT, 1 mM ATP), 1 μl of T4 DNA ligase was added, and the total amount was made up to 20 μl with distilled water.
Incubated for 12 hours. Next, Escherichia coli JM103 was subjected to the calcium chloride method (Journal of Mole
LB plate medium containing 20 μg / ml of chloramphenicol (1% (W /
V) Bacot-trypton, 0.5% (W / V) Bacto-yeast extrac
Chloramphenicol resistant colonies were selected at (t, 1% (W / V) NaCl and 1.5% (W / V) agar). Plasmid DNA from chloramphenicol-resistant clones
And a clone containing a fragment of about 4.0 kb derived from plasmid pNL4-3 DNA was selected by Hind III digestion, and cloned.
pNLH402 was obtained.

5 μl of plasmid pNLH402 DNA (5 μg)
Of Hind III and 10 μl of 5 × RM, and the whole amount is
After incubating at 37 ° C. for 1 hour, phenol extraction and chloroform treatment were performed, followed by ethanol precipitation. 10 μl of 5 × RM and 5 μl of Bgl II were added to the precipitate, and the whole amount was dissolved in distilled water to 50 μl, and the mixture was dissolved well at 37 ° C., incubated again at 37 ° C. for 1 hour, and subjected to phenol extraction and chloroform treatment, followed by ethanol precipitation of 10 μl. Dissolved in TE.

On the other hand, the expression vector pUR290 (The EMBO Journal, 2
(2): 1791-1794,1983) and 5 μl of Hind II.
I, 10 μl of 5 × RM was added, the mixture was made up to 50 μl with distilled water, incubated at 37 ° C. for 1 hour, subjected to phenol extraction, chloroform treatment, ethanol precipitation, and 5 ×
10 μl of RM (NaCl concentration is 500 mM) and 5 μl of BamHI,
Further, 35 μl of distilled water was added to completely dissolve the precipitate, and the mixture was again incubated at 37 ° C. for 1 hour, and after phenol extraction and chloroform treatment, 10 μl of DNA precipitated with ethanol was added.
Was dissolved.

Next, pUR290 (1) cut with Hind III and BamHI described above.
μl) and cut with Hind III and Bgl II. pNLH402 (1
μl), 2 μl of 10 × ligation buffer and 1 μl of T4 DNA ligase were added, and the whole volume was distilled water.
μl and incubated at 15 ° C. for 12 hours. E. coli UT481 strain (Journal of Bacteriology, 16
3: 376-384, 1985) by the calcium chloride method described above, ampicillin-resistant colonies were selected on an LB plate medium containing 20 μg / ml of ampicillin, and a 3.8 kb fragment derived from pNL4-3 was further isolated. The clones containing the clone UT48 were selected by measuring the size of the inserted fragment by EcoRI digestion.
1 / pPG280 was obtained. That is, this clone was constructed using plasmid pUR2
A 3.8 kb HIV pol gene region is linked to the 3 'end of the lacZ gene on 90, and the lacZ and pol gene products are expressed as a fusion protein (about 230 kd) and then processed to produce various enzymes. It is estimated that.

Example 2 Production of Lentiviral Protease, Reverse Transcriptase, and Integrase Enzymes by Culture of Transformants:
Transformant clone UT481 / pPG280 was added to ampicillin 20 μg /
ml of LB medium (Bacto-tryptone 1% (W / V), Bacto-
yeast extract 0.5 (W / V) and NaCl 1% (W / V)
After culturing at 37 ° C. for 18 hours, 1/100 volume was added to a fresh LB medium (containing 20 μg / ml of ampicillin) and pre-cultured at 25 ° C. When the turbidity OD ₆₀₀ nm of the medium reaches 0.5, IPTG (Is
opropyl-thiogalactoside, Sigma [USA]) 1mM
In addition, the culture was further continued at 25 ° C. for 18 hours. Centrifugation operation (50
(00 rpm, 5 minutes) to collect the cells, 1/25 volume of 40 mM Tris-HC
l, pH 8.0 (0.1 mM EDTA, 5 mM MgCl ₂ , 0.1% (W / V) TritonX
Containing -100 and 10 mM 2-mercaptoethanol)
And ultrasonic treatment (5 treatments for 30 seconds, 19.5KHz, 300
After W), the supernatant was separated by centrifugation (19,000 rpm, 60 minutes). FIG. 1 shows the results of measuring the reverse transcriptase activity of the crude extract in order to examine the HIV pol gene product in the washed extract. Expected significant reverse transcriptase activity was observed. In addition, analysis by Western blotting was also performed. That is, 4% (W / V) sodium dodecyl sulfate (SDS) and 1% (W / V) 2-mercaptoethanol were added to the collected cells, and the mixture was boiled for 5 minutes and centrifuged (10,000 rpm,
The resulting supernatant was subjected to electrophoresis using a gel of 0.1% (W / V) SDS-10% (W / V) polyacrylamide, and was subjected to transblot cell (Bio-Rad, USA). After blotting on a nitrocellulose membrane (manufactured by S & S [West Germany]), the membrane was blocked by immersion in a 3% (W / V) gelatin solution as usual. Next, the filter was subjected to a primary reaction with an HIV carrier serum, washed, and then subjected to a secondary reaction with a peroxidase-labeled goat anti-human IgG serum (Bio-Rad, USA). Finally, after washing the filter, 0.4 ml of DAB (3,3'-d) was added to 50 ml of TBS (20 mM Tris-HCl, pH 7.4, 500 mM NaCl).
iaminobenzidine tetrahydrochloride) and 30% (W / V)
Was immersed in a coloring solution to which 15 μl of a hydrogen peroxide solution was added, and the color was developed at room temperature for 15 minutes. The filter was washed with distilled water to stop the reaction. FIG. 2 shows the results. Transformant UT481 / pUR290 by vector pUR290 without HIV pol gene
No specific band reacting with the HIV carrier serum was observed in the crude extract of UT481 / pPG280, but in the crude extract of UT481 / pPG280, reverse transcriptase of molecular weight 66 kd and 51 kd, which is the HIV pol gene product, and 32 kd Integrase and 12 kd protease bands could be seen. Reverse transcriptase
The fact that it is cleaved from galactosidase can be easily seen from the results of column chromatography of the anion exchanger MonoQ (Pharmacia [Sweden]) in FIG. That is, the reverse transcriptase activity was observed in the fraction completely separated from the β-galactosidase activity. Therefore, HIV
Although the pol gene product is produced as a fusion protein with β-galactosidase, the protease, reverse transcriptase and integrase regions are specifically cleaved by the pol gene product's protease and accumulate in the cells. Inferred.

Example 3 Construction of a Vector Producing Lentiviral Protease in Large Amount: The pol Gene Expression Plasmid p Obtained in Example 1
5 μg of PG280 DNA, 5 μl of Hind III, 20 μl of 5 × RM
Was added to make a total volume of 100 μl with distilled water, incubated at 37 ° C. for 1 hour, extracted with phenol, treated with chloroform, and precipitated with ethanol. Add 5 μl Bal I and 10 μl
5 × RM (-NaCl) was added, and the whole amount was dissolved in distilled water to 50 μl, well dissolved, incubated again at 37 ° C. for 1 hour, extracted with phenol, treated with chloroform, and precipitated with ethanol. 5 μl of 10 × polymerase buffer (670 mM)
M Tris _{· HCl, pH8.8,67mM MgCl 2, 166mM} (NH 4) 2 SO 4, 100
mM 2-mercaptoethanol, 67 mM EDTA) and 5 μl of 10
× NTP solution (3.3 mM dATP, dGTP, dTTP and dCTP each) and 1
μl of T4 DNA polymerase and add 50 μl with distilled water.
The mixture was well dissolved as 1 and incubated at 37 ° C. for 15 minutes, extracted with phenol, treated with chloroform, and precipitated with ethanol. This precipitate was dissolved in 10 μl of TE, and 2 μl was added to 1 μl.
1 μl of 10 × Igation buffer, 1 μl
T4 DNA ligase was added, and the total volume was adjusted to 20 μl with distilled water, followed by incubation at 15 ° C. for 12 hours. Escherichia coli UT481 strain was transformed with this reaction solution by the above-mentioned calcium chloride method, ampicillin-resistant colonies were selected on an LB plate medium containing 20 μg / ml of ampicillin, and further, pNL4-3-derived 0.
A clone containing the 55 kb fragment was selected by measuring the size of the inserted fragment by EcoR I digestion, and clone UT481 / pL
B550-3 was obtained.

Construction of a vector producing a large amount of HIV-1 (lentivirus) protease: pNLH402 (Example 1) was replaced with Bgl II
And Kpn I, and the resulting DNA fragment of about 1.7 kb was recovered and further cut with Nla IV. About 1.2 kb of DNA generated at this time
The fragment was inserted and ligated into the Hinc II site of the cloning vector pUC19 to obtain pUN40. pUN40 was digested with BamH I and Pst I, and the resulting DNA fragment of about 1.2 kb was digested with BamH I and Pst I into an expression vector pUR291 (The EMBO Journal, 2).
(2): 1791-1794, 1983) and pT7-7 (Proc. Natl. Aci.
d. Sci. USA, 82: 1074-1078, 1985), pPG401 and
pTP440 was constructed respectively. pPG401 was digested with Bal I and Pst I, treated with T4 DNA polymerase, and then self-ligated to obtain pPG421. In addition, pTG440 was replaced with Bal I and Pst
After digestion with I, treatment with T4 DNA polymerase and self-ligation, pTP442 was obtained. pPG401, pPG421, pTP440 and pTP442 express the HIV-1 protease. pPG401
And pPG421, UT481 strain and JM103 strain were used as expression hosts. In pTP440 and pTP442, BL was used as the host for expression.
21 (DE3) strain was used.

Example 4 Large-scale production of lentivirus protease by transformant: After culturing transformant clone UT481 / pLB550-3 in LB medium (containing 20 μg / ml of ampicillin) at 37 ° C. for 18 hours, 1/100 volume was freshened. It was added to an LB medium (containing 20 μg / ml of ampicillin) and precultured at 37 ° C. Medium turbidity OD ₆₀₀ nm
Reached 0.5, IPTG (Isopropyl β-D-Thiogal
Actoside, Sigma (USA)) was added at 1 mM, and the culture was continued at 37 ° C. for 6 hours. Collect cells by centrifugation (500 rpm, 5 minutes), 4% (W / V) sodium dodecyl sulfate (SDS)
And 1% (W / V) of 2-mercaptoethanol,
The mixture was boiled for 10 minutes, centrifuged (10,000 rpm, 5 minutes), and the supernatant was subjected to electrophoresis using a gel of 0.1% (W / V) SDS-15% (W / V) polyacrylamide. The analysis was performed by Western blotting as described in the above section. UT481 / pU
No specific band reacting with the HIV carrier serum was observed in the crude extract of R290, but a 12 kd protease band was observed in the crude extract of UT481 / pLB550-3. The amount of protease produced by pLB550-3 was pPG2
It was a multiple of 80. This clone is the plasmid pUR290
A 0.55 kb HIV pol gene is ligated to the 3 'end of the lacZ gene, and the lacZ-pol gene product is produced as a fusion protein with a molecular weight of about 140 kd, and then processed to produce a protease with a molecular weight of about 12 kd It is estimated to be.

Example 5 Construction of an expression vector in which an oncovirus protease gene and a pol gene are inserted and linked: Avian sarcoma virus DNA clone pSRA2 (Journal of virology, Vol. 36, No. 5)
0-61, 1980) 5 μg of BamHI and 2 μl of DNA
0 μl of 5 × RM is added, and the total volume is adjusted to 100 μl with distilled water.
Incubated for 1 hour at ° C. 1% of this reaction solution
(W / V) 1.8 kb DNA after electrophoresis on low melting point agarose gel
The gel containing the DNA fragment was cut out, extracted with phenol, treated with chloroform, and precipitated with ethanol. The DNA precipitate was dissolved in 10 µl of TE, and 1 µl of the plasmid pUR291 which had been cleaved with BamHI and treated with alkaline phosphatase.
1 μl (0.1 μl) of DNA, 2 μl of 10 × ligation buffer and 1 μl of T4 DNA ligase were added, and the total volume was adjusted to 20 μl with distilled water, followed by incubation at 15 ° C. for 12 hours. Next, Escherichia coli UT481 strain was transformed with the reaction solution by the calcium chloride method, and ampicillin 20 μg / ml was used.
Ampicillin-resistant colonies were selected on the contained LB plate medium. Plasmid DNA was extracted from the ampicillin-resistant clone by a conventional method, and a clone containing a 1.8 kb fragment derived from the plasmid pSRA2 and producing a lacZ-gag fusion protein was selected to obtain pSR281.

5 µg of plasmid pSRA2 DNA and 5 µl of Pst I and 20 µl
Of 5 × RM (750 mM NaCl) and add 100 μl with distilled water
and incubated at 37 ° C for 1 hour. This reaction solution was subjected to 1% (W / V) low-melting point agarose gel electrophoresis and then subjected to 3.1%
Cut out gel containing kb DNA fragment, extract with phenol,
After chloroform treatment, the precipitate was precipitated with ethanol and dissolved in 10 μl of TE. Similarly, M13mP18 double-stranded phage DNA was
The fragment was cleaved with I and treated with alkaline phosphatase. To 1 μl (0.1 μg) of this DNA, 1 μl of the above 3.1 kb DNA fragment, 2 μl
1 × 10 × ligation buffer, 1 μl of T4 DNA trigger, and make up to 20 μl with distilled water.
Incubated for hours. Next, using this reaction solution, phage DNA was added to E. coli TG1 strain by the calcium chloride method.
And transfected with X-gal (5-bromo-4).
-Chloro-3-indolyl-β-D-galactopyranoside,
Sigma (USA) 2YT plate medium (1.6 µg / ml)
% (W / V) Bacto-trypton, 1% (W / V) Bactoyeast extra
ct, 0.5% (W / V) NaCl and 1.5% (W / V) Bacto-agar)
To form plaques. Next, the TG1 strain was transformed into a 2YT liquid medium (1.6% (W / V) Bacto-trypton, 1% (W / V) Bacto-yeas).
t extract, 0.5% (W / V) NaCl), grown to a turbidity OD ₆₀₀ nm = 0.3, inoculated with several colorless clones among the resulting plaques, and continued culturing for several hours. Single chain
DNA and double-stranded DNA were prepared respectively. By cutting the obtained double-stranded input DNA with Pst I and BamHI, pS
A clone M13sr31 containing a 3.1 kb fragment derived from RA2 was selected. The 3.1kb fragment derived from pSRA2 contains gag (protease)
The 3 'end of the gene, its stop codon TAG and the pol gene are encoded, and if one base is inserted before the stop codon, a gag-pol fusion gene having the same translation frame can be obtained.

The authors obtained an ATAG-modified clone M13sr32 by inserting one base before the TAG stop codon on M13sr31 using an in vitro mutagenesis kit from Amersham.

To 5 μg of M13sr32 double-stranded DNA, 5 μl of Pst I and 20 μl of 5 × RM were added, and the total volume was made up to 100 μl with distilled water, followed by incubation at 37 ° C. for a long time. This reaction solution was subjected to 1% (W / V) low-melting point agarose gel electrophoresis, followed by cutting out a gel containing a DNA fragment of 3.1 kb, phenol extraction, chloroform treatment, and ethanol precipitation. The DNA precipitate was washed with 10 μl of T
1 μl (0.1 μl) of plasmid pSR281 DNA digested with Pst I and treated with alkaline phosphatase
μg), then 2 μl of 10 × ligation buffer, 1 μl
Was added, and the total volume was adjusted to 20 μl with distilled water, followed by incubation at 15 ° C. for 12 hours. Next, Escherichia coli UT481 strain was transformed with this reaction solution by the calcium chloride method, and ampicillin-resistant colonies were selected on an LB plate medium containing 20 μg / ml of ampicillin. Plasmid DNA was extracted from the ampicillin-resistant clone by a standard method,
By cutting with stI and BamHI, the presence and direction of the 3.1 kb fragment derived from M13sr32 was confirmed, and clone UT481, in which the expression of protease and pol gene products was expected, was confirmed.
/ pSR271 was obtained. In addition, 1.8k derived from pSRA2 contained in pSR281
1.3 kb overlapping with the 3.1 kb fragment from M13sr32 in the b region
Is cut out when pSR281 is cut with PstI.

Example 6 Production of oncovirus protease, reverse transcriptase, and integrase enzymes by culturing the transformant;
After culturing the transformant clone UT481 / pSR271 in LB medium (containing 20 μg / ml of ampicillin) at 37 ° C. for 18 hours, 1/100 volume was added to fresh LB medium (containing 20 μg / ml of ampicillin),
Precultured at 25 ° C. When the suspension OD ₆₀₀ nm of the medium reached 0.5, 1 mM of IPTG was added, and the culture was further continued at 25 ° C. for 18 hours. The cells were collected by centrifugation (5000 rpm, 5 minutes), and 1/25 volume of 40 mM Tris-HCl, pH 8.0 (0.1 mM EDTA, 5 mM MgCl ₂ , 0.
After suspending in 1% (W / V) Triton X-100 and 10 mM 2-mercaptoethanol, sonication (5 treatments for 30 seconds, 19.5 KHz, 300 W), and centrifugation (19,000 rpm, 60 minutes) )
Was used to separate the supernatant. In order to examine the RSV gene product in the crude extract, the reverse transcriptase activity of the crude extract was measured.
In addition, analysis by Western blotting was also performed. That is, 4% (W / V) sodium dodecyl sulfate (SDS) and 10% (V / V) 2-mercaptoethanol were added to the collected cells, and the mixture was boiled for 5 minutes and centrifuged (10,000 rpm, 5 rpm). The resulting supernatant was subjected to electrophoresis using a gel of 0.1% (W / V) SDS-15% (W / V) polyacrylamide, and subjected to nitrification using a trans-blot cell (Bio-Rad, USA). After blotting on a cellulose membrane (manufactured by S & S [West Germany]), the membrane was immersed in a 3% (W / V) gelatin solution and blocked as usual. Next, the filter was subjected to a primary reaction with anti-RSV rabbit serum, washed, and then subjected to a secondary reaction with peroxidase-labeled goat anti-rabbit IgG serum (Bio-Rad, USA). Finally, after washing the filter, 50 ml of TBS (20 mM Tris-HCl, p
0.4 ml of DAB (3,3'-diaminobenzi) in H7.4,500 mM NaCl
(dine tetrahydrochloride) and 15 μl of a 30% (W / V) hydrogen peroxide solution, and the mixture was immersed for 15 minutes at room temperature, and the filter was washed with distilled water to stop the reaction. RS
Transformant U by vector pUR290 without V gene
No specific band reacting with anti-RSV rabbit serum was found in the crude extract of T481 / pUR290, but a reverse transcriptase (p92, p65) band of RSV was observed in the crude extract of UT481 / pSR271. I was able to do it. The RSV protease and pol gene products are produced as fusion proteins with β-galactosidase, but the protease, reverse transcriptase and integrase regions are specifically cleaved by the gag gene product protease and accumulate in the bacterial cells. It is presumed that it will be done. In addition, clone UT481 / pSR271
3.6 kb Rous sarcoma virus g at the 3 'end of the cZ gene
The ag and pol gene regions are linked, and the lacZ, gag and pol gene products are expressed as a fusion protein (approximately 230 kd) and then processed to produce protease (p15), reverse transcriptase (p92, p65), integrase (P32) is presumed to be produced.

Example 7 Extraction of reverse transcriptase: As described in Example 2, transformed E. coli clone UT481 / pPG280 was cultured at 25 ° C. in 9 LB medium (containing 20 μg / ml of ampicillin) to obtain the turbidity OD of the bacteria.
_{When 600} nm reaches 0.5, add 1 ml of IPTG,
Continue culturing for hours, and after collecting the cells, 120 ml of 40 mM Tris-HCl, pH
8.0 (containing 0.1 mM EDTA, 5 mM MgCl ₂ , 0.1 W / V Triton X-100 and 10 mM 2-mercaptoethanol) buffer, disrupt cells by sonication, and centrifuge (1
9,000 rpm for 60 minutes), and the supernatant was separated as a crude extract.

Example 8 Crude purification of reverse transcriptase: 0.1% (W / V) of polymin P (manufactured by BRL [USA]) was added to the crude extract, the mixture was stirred at 4 ° C for 30 minutes, and centrifuged (16,000 rpm, 20 Min), the ammonium sulfate solution was added to the resulting supernatant, and the precipitate formed as a 40% saturation was removed by centrifugation (16,000 rpm, 20 minutes).
I got Again, an ammonium sulfate solution was added to 80% saturation, and the resulting precipitate was dissolved in the above 50 ml of 40 mM Tris / HCl buffer and dialyzed against the same buffer containing 50 mM NaCl.

Example 9 Purification of reverse transcriptase: Next, purification was performed using DEAE Biogel A (manufactured by Bio-Rad [USA]) and Affigel Heparin column chromatography (manufactured by Bio-Rad). 40 mM Tris-HCl, pH 8.0 (0.1 mM EDTA, 5 mM MgCl ₂ , 0.1% (W / V) Tr
itonX-100, 10 mM 2-mercaptoethanol and 50 mM
After passing the dialyzed sample through a 30 ml DEAE Biogel A column equilibrated with a buffer (containing NaCl), the solution was passed through a 30 ml Affigelheparin column equilibrated with the above buffer, and sodium chloride was added. Gradient 50mM ~ 400mM
Was eluted with 150 ml of eluate. The results are shown in FIG. Fractions 29-38 containing reverse transcriptase activity were pooled. The reverse transcriptase solution obtained by pooling was 20 mM sodium phosphate buffer, pH 6.8.
(Containing 0.1 mM EDTA, 5 mM MgCl ₂ , 0.1% (W / V) Triton X-100 and 10 mM 2-mercaptoethanol), and a hydroxyapatite column (KB column, manufactured by Koken [Japan]) And further purified by high performance liquid chromatography. That is, after the above dialyzed sample was adsorbed on the column, elution was performed with a continuous concentration gradient of 20 to 400 mM sodium phosphate, and fractions containing reverse transcriptase activity were pooled to obtain purified reverse transcriptase. The purity of this purified enzyme is SD
S-PAGE confirmed that it was 95% (W / W) or more,
The yield was 31% (W / W) based on the crude extract. The purified reverse transcriptase was composed of almost equimolar ratios of p64 and p51, and when the N-terminal amino acid sequences of both were determined, Pr
o-Ile-Ser-Pro-Ile-Glu-Thr-Val-Pro-Val-Ly
s-Leu-Lys-Pro-Gly -... ‥, which coincided with the amino acid sequence predicted from the nucleotide sequence.

Example 10 Construction of Vector Expressing AIDS Virus Core Protein in Large Quantity: Plasmid pNL4-3 carrying HIV-1 provirus genomic DNA (Journal of Virology, 59 (2): 284-
291,1986) is cut with the restriction enzyme Pvu II, and
The DNA fragment was recovered. This fragment was ligated with Hinc of plasmid pUC9.
Inserted at the II site. And the 5 'end of the inserted fragment is Ba
The DNA linked to the mHI site was designated pCV91.

After cutting pCV91 with BamHI and BalI, the resulting ~ 1.5k
The DNA fragment of b is recovered, and Sal I, T4 DNA polymerase, and B
Expression vector pUR292 (The EMBO Jo
urnal, 2 (2): 1791-1794, 1983).
Was prepared. Furthermore, pPG912 was cut with BglII, treated with T4 DNA polymerase, and then self-ligated to prepare pPG922 having 4 bases inserted into the BglII site of pPG912.

In pPG912 and pPG922, the 3 'of the lacZ gene on pUR292
At the end, a DNA fragment containing the entire region from the p24 region of the HIV-1 gag gene to the protease region of the pol gene was in-frame.
Are connected by The gene product of HIV-1 is expressed as a fusion protein with β-galactosidase, followed by pPG912
In pPG922, p24 core protein is produced through a process by a protease that is expressed via frame shifting from gag to pol, and in pPG922, without frame shifting. Note that pPG912 also produces p15.

The above expression plasmids pPG912 and pPG922 were replaced with BamHI and Cl.
After digestion with aI, the resulting DNA fragments of about 1.5 kb were respectively recovered, and the expression vector pT7-7 cleaved with BamHI and ClaI was obtained.
(Proc. Natl. Acid. Sci. USA, 82: 1074-1078, 1985) to prepare pTG11 and pTG12. Next, pTG11 and pTG12
After cleavage with BamHI and treatment with T4 DNA polymerase,
By self-ligation, pTG110 and pTG120 were prepared. this is,
The same HIV protein region as the derived pPG912 and pPG922 is expressed as a fusion protein with an oligopeptide derived from pT7-7 under the control of the T7 promoter. The fusion protein undergoes a protease process, resulting in the production of p24 and p15.

In addition, E. coli JM103 strain (Nuc
leic Acid Research, 9 (2): 309-321, 1981), pPG912
Escherichia coli UT481 strain (J
ournal of Bacteriology, 163: 376-384,1985), pTG110
And E. coli BL21 (DE as a host for protein expression in pTG120)
3) Strain (Journal of Molecular Biology, 189 (1): 113)
−130, 1986). The yields of purified p24 and p15 per E. coli culture are shown in Table 1.

Example 11 Construction of a vector that expresses a large amount of the nucleoprotein of AIDS virus: The gag-pol gene expression plasmid pPG912 prepared in Example 10 was cut with Hind III, and the resulting plasmid was expressed at about 0.1 μg.
A 9 kb DNA fragment was recovered. Next, this fragment was used to express the expression vector pUR290 (The EMBO Journal, 2 (2): 1791-1794,198).
The plasmid pPG930 inserted in the Hind III site of 3) in a preferred direction was prepared. In pPG930, polZ was added to the 3 'end of the lacZ gene on pUR290 from the p15 region of the HIV-1 gag gene.
A DNA fragment completely containing the protease region of the gene is i
They are linked by an n-frame. The HIV gene product is expressed as a fusion protein with β-galactosidase. This fusion protein is processed by a protease expressed through frame shifting, resulting in the production of p15 nucleoprotein.

In addition, the above 0.9 kb Hind III fragment was expressed in the expression vector pT7.
Plasmid pTG21 inserted in the HindIII site of -7 in the preferred orientation was constructed. Furthermore, pTG21 is cut with Sal I, T4 DNA
After the polymerase treatment, pTG2
I got 10. pTG210 expresses the same HIV protein region as pPG930 as a fusion protein with an oligopeptide derived from pT7-7 under the control of the T7 promoter. This fusion protein is processed by a protease expressed through frame shifting, resulting in the production of p15.

The JM103 strain was used as a plasmid construction host, the UT481 strain was used as a host for protein expression in pPG930, and the BL21 (DE3) strain was used as a host for protein expression in pTG210. The yield of purified p15 per E. coli culture is shown in Table 1. Table 1 Yield of purified protein per culture solution Expression plasmid Host p24 (mg) p15 (mg) pPG912 UT481 5 0.7 pPG922 UT481 5-pPG930 UT481-1 pPG110 BL21 (DE3) 193 pPG120 BL21 (DE3) 10- pPG210 BL21 (DE3) -5 When the N-terminal amino acid sequences of purified p24 and p15 were determined, p24 was found to be Pro-Ile-Val-Gln-Asn-Leu-Gln-Gl.
y-Gln-Met-Val-His-Gln-Ala-Ile -..., p15 is Il
e-Gln-Lys-Gly-Asn-Phe-Arg-Asn-Gln-Arg-Ly
s-Thr-Val-..., each of which coincided with the amino acid sequence predicted from the nucleotide sequence.

Example 12 AIDS virus core protein p2 from culture of transformant
Extraction 4: Transformant clone UT481 / pPG922 in LB medium containing 20 μg / ml ampicillin (Bacto-tryptone 1% (W /
V), Bacto-yeast extract 0.5% (W / V) and NaCl 1%
(W / V)) at 37 ° C for 18 hours, 1/100 volume of fresh LB
The medium was added to a medium (containing 20 μg / ml of ampicillin) and pre-cultured at 37 ° C. When the turbidity OD _{600nm of the} medium reaches 0.5, IPTG
(Isopropyl-thiogalactoside: Sigma [USA] 1
mM was added, and the culture was continued at 37 ° C. for 8 hours. The cells were collected by centrifugation (5,000 rpm, 10 minutes), suspended in 1/50 volume of 50 mM sodium phosphate pH 7.0, sonicated (5 times 30 seconds, 19.5 kHz 300 W), and then centrifuged (19,000 rpm, 60 minutes)
To separate the supernatant.

Rough purification of p24: Ammonium sulfate was added to the crude extract so as to be 35% saturated, stirred, and centrifuged (16000 rpm, 20
) Was dissolved in 30 ml of 20 mM sodium malonate, pH 5.3, and dialyzed against the same solution.

Purification of p24: The dialyzed sample was treated with 20 mM sodium malonate p
Pass through a MonoS column (manufactured by Pharmacia [Sweden]) equilibrated with H5.3 and run a sodium chloride gradient (0 to 1).
M). Fractions containing p24 were pooled and dialyzed against 20 mM Tris-HCl buffer pH 8.4. After dialysis, the solution is passed through a MonoQ column (Pharmacia [Sweden]) equilibrated with the same buffer, and a sodium chloride gradient (0 to 1 M) is used.
To give purified p24.

Example 13 Extraction of p15: Transformant clone BL21 (DE3) / pTG210
Was cultured at 37 ° C. for 18 hours in an LB medium containing 20 μg / ml of ampicillin. To the fresh LB medium (containing 20 μg / ml ampicillin), the above-mentioned culture solution was added at 1/100 volume equivalent, and cultured at 37 ° C. When medium turbidity OD _600nm reached 0.5, IPTG is added to a 1 mM, was further incubated for 5 hours at 37 ° C.. The cells are collected by centrifugation (5000 rpm, 10 minutes), suspended in 1/50 volume of the culture solution, 20 mM sodium phosphate pH 7.0, and subjected to ultrasonic treatment (6 times of 30 seconds, 19.5 kHz, 300 W). , Centrifugal operation (19000
(rpm, 60 minutes) to separate as a crude extract.

Rough purification of p15: Ammonium sulfate was added to the crude extract to 60% saturation, stirred, and centrifuged (16000 rpm, 20
Min). The resulting precipitate is washed with 20 mM sodium phosphate pH
It was dissolved in 7.0 and dialyzed with the same solution.

Purification of p15: dialyzed sample is 20 mM sodium phosphate pH
It was passed through a phosphocellulose column (Whatman [UK]) equilibrated in 7.0. Eluted with a 0-1 M sodium chloride gradient, pooled fractions containing p15 and dialyzed against 20 mM sodium phosphate pH 7.0. This is passed through a hydroxyapatite column (KB column, manufactured by Koken Co., Ltd.),
Elution was performed with a 20-700 mM sodium phosphate gradient. Fractions containing p15 were pooled and dialyzed again against 20 mM sodium phosphate, pH 7.0. This sample was passed through a MonoS column and eluted with a 0-1 M sodium chloride gradient. Fractions containing p15 were pooled to give purified p15.

Example 14 Construction of an expression plasmid into which the gag-pol gene of AIDS virus was inserted and ligated: HIV-1 provirus DNA clone p
NL4-3 was digested with Hind III, and the resulting DNA fragment of about 1.2 kb was cloned into HindIII of cloning vector pHSG398 (Takara Shuzo)
Insertion at the III site created pNLH122. pglH122 to Bgl I
Cleavage with I and Hind III, the resulting DNA fragment of about 1.03 kb
M13mp19 (Takara Shuzo) DNA cleaved with mHI and Hind III
Was inserted and ligated to prepare Gag19 · (Bg-H). oligo
nucleotide-directed in vitro mutagenesis system V
Using ersion2 (manufactured by Amersham [UK]), Gag19 · (Bg
-H) The nucleotide sequence GAG immediately before the translation initiation codon (ATG) of the above gag gene was modified to CAT, and G was newly introduced into the NdeI site.
ag19 · (Nde) was prepared. Gag19 · (Nde) is converted to Nde I and
After digestion with Pst I, the resulting DNA fragment of 0.36 kb was digested with Nde I and Pde I.
It was inserted and ligated into pT7-7 cleaved with stI to prepare pTG541. Furthermore, pPG912 and pPG922 produced in Example 10 were
Cleavage at tI, the resulting DNA fragments of about 1.2 kb were
Inserted into Pst I site of 1 and ligated in the preferred direction
These were pTG591 and pTG592. In pTG591 and pTG592, pol from the translation initiation codon of the gag gene under the control of the T7 promoter
A DNA fragment completely including the protease region of the gene is ligated. In pTG591, the expressed HIV protein is processed by a protease expressed through frame shifting, and a large amount of p17, p24 and p15 is produced.
The HIV protein expressed in pTG592 is subjected to a process by a protease that is expressed without going through frame shifting, and a large amount of p17 and p24 is produced.

The JM103 strain was used as a host for plasmid construction, and the BL21 (DE3) strain was used as a host for protein expression.

Example 15 Construction of a vector expressing a large amount of the AIDS virus matrix protein: pTG591 prepared in Example 14 was cut with NsiI and ApaI, treated with S1 nuclease, and then self-ligated. N-terminal side of NagI site of gag gene and Ap
When the C-terminal side of the aI site is fused in-frame, p2
Gag protein and Gag-Po lacking from the middle of 4 to the middle of p15
It is presumed that p17 is produced by the expression of l proteins, which are processed by the latter protease. P55 Gag from many clones obtained by the above operation
A clone expressing the activity of processing a protein and producing a large amount of p17 was selected and named pTG691.

pTG591 was digested with BglII, treated with T4 DNA polymerase, and further digested with PstI. About 0.52 kb D generated at this time
The NA fragment was recovered. On the other hand, pTG591 was sequentially treated with NsiI, S1 nuclease, and PstI, and a DNA fragment of about 2.9 kb generated at this time was recovered. The two DNA fragments were ligated to obtain a large number of clones. Among them, a clone expressing the activity of processing the p55 Gag protein and producing a large amount of p17 was selected and named pTG681.

pTG591 was digested with Sph I and Apa I, treated with T4 DNA polymerase, and self-ligated to prepare pTG171.

pTG592 was cut with Sph I and Apa I, treated with T4 DNA polymerase, and self-ligated to prepare pTG172.

Gag19 • (Nde) prepared in Example 14 was replaced with EcoR I and Pst
I, and the resulting DNA fragment of 0.76 kb was ligated with EcoR I and Pst
Inserted and ligated into M13mp18 cleaved with I, Gag18- (Bg-
P) was prepared. Gag18 ・ in vitro mutagenesis
(Bg-P) The gag gene on the gag gene, CCT at the 133rd position was modified to TAA, and Gag18 • TAA was prepared in which a stop codon was inserted immediately after the region encoding p17. Gag18TAA with Nde I and P
Cleavage with stI, the resulting 0.63 kb DNA fragment was digested with NdeI and Pst
It was inserted and ligated into pT7-7 cleaved with I to prepare pTG52.

Defective Gag proteins and Gag by proteases expressed via frame shifting in pTG691 and pTG171 and without frame shifting in pTG681 and pTG172.
-The Pol protein undergoes processing, producing large amounts of p17.
Also, pTG52 does not need to undergo post-translational processes.
Directly in large quantities.

The JM103 strain was used as a host for plasmid construction, and the BL21 (DE3) strain was used as a host for protein expression.

Example 16 Extraction of p17: Transformant clone BL21 (DE3) / pTG171
Was cultured at 37 ° C. for 18 hours in an LB medium containing 20 μg / ml of ampicillin. To the fresh LB medium (containing 20 μg / ml ampicillin), the above-mentioned culture solution was added at 1/100 volume equivalent, and cultured at 37 ° C. When medium turbidity OD _600nm reached 0.5, IPTG is added to a 1 mM, was further incubated for 5 hours at 37 ° C.. The cells were collected by centrifugation (5000 rpm, 10 minutes), suspended in 1/50 volume of the culture solution, 15 mM sodium phosphate, pH 6.7, and sonicated (30 seconds, 6 times, 19.5 kHz, 300 W). , Centrifugal operation (19000
(rpm, 60 minutes) to separate as a crude extract.

Rough purification of p17: Ammonium sulfate was added to the crude extract to 40% saturation, and the mixture was stirred and centrifuged (16000 rpm, 20 minutes)
Was done. Ammonium sulfate was added to the obtained supernatant so as to be 80% saturated, and the mixture was stirred and centrifuged (16000 rpm, 20 minutes). The obtained precipitate was dissolved in 15 mM sodium phosphate, pH 6.7, and dialyzed against the same solution.

Purification of p17: dialyzed sample is 15 mM sodium phosphate pH
It passed through S-sepharose (Pharmacia [Sweden]) equilibrated in 6.7. Eluted with a 0-1 M sodium chloride gradient, pooled the fractions containing p17 and diluted 2-fold with 15 mM sodium phosphate pH 6.7. This was passed through a MonoS column (Pharmacia) equilibrated with the same solution. 0
Eluted with a ~ 1 M sodium chloride gradient, pooled fractions containing p17, 20 mM sodium phosphate pH 7.0, 10 mM 2-
It was diluted 5-fold with mercaptoethanol. This was passed through a hydroxyapatite column (KB column, manufactured by Koken Co., Ltd.), and eluted with a sodium phosphate gradient of 15 to 700 mM. Approximately 4mg from one transformant culture by this method
Was obtained. The purified p17 had the N-terminal methionine residue removed, but had not undergone myristoylation, and had an N-terminal amino acid sequence of Gly-Ala-Arg-A
la-Ser-Val-Leu-Ser-Gly-Gly-Glu-Lre-Asp-L
ys-Trp... agreed with the amino acid sequence predicted from the nucleotide sequence.

Example 17 Construction of Expression Plasmid into Which HIV-1 Nef Gene was Inserted and Ligation: HIV-1 Provirus DNA Clone pNL4-3
The fragment was cleaved with dIII, and the resulting DNA fragment of about 1.5 kb was inserted into the HindIII site of the cloning vector pHSG398 to prepare pNLH152. pNLH152 was digested with Hind III and Xho I, and the resulting 0.72 kb Xho I-Hind III fragment was digested with Sal I and Hi.
Expression vector pUR292 cleaved with nd III (The EMBO Journal
al, 2 (2): 1791-1794, 1983).
Was prepared. pNF102 was digested with BamHI and HindIII, and the resulting 0.72 kb BamHI-HindIII fragment was digested with BamHI and HindIII.
Insert and ligate into the expression vector pT7-7 cleaved with III
TF103 was produced. pTF103 was cleaved with BamHI, treated with T4 DNA polymerase, and self-ligated to produce pTN104. pNF102 expresses a large amount of a chimeric protein consisting of β-galactosidase and a region from amino acids 35 to 206 (C-terminal) of HIV-1 Nef protein. pTN104 is pT7 at the N-terminus of the region from amino acids 35 to 206 of the Nef protein.
It expresses a large amount of a chimeric protein to which a peptide consisting of 11 amino acids derived from the multiple cloning site of -7 has been added.
These chimeric proteins were tested for HIV by Western blotting.
-1 Reacted specifically with infected sera. As a host for expression, the JM103 strain and the UT481 strain were used for pNF102, and the BL21 strain was used for pTF104.
(DE3) strain was used.

Example 18 Construction of Vector Expressing Nef Protein of HIV-1 in High Level: p
NLH152 was cut with Hind III and Hinc II, resulting in approximately 0.96 k
The Hinc II-Hind III fragment of b was inserted and ligated into M13mp19 cleaved with Hinc II and Hind III to prepare Nef19 · (Hc-H). By in vitro mutagenesis, Nef19 · (Hc-
H) Nef obtained by modifying the nucleotide sequence AAG immediately before the translation initiation codon (ATG) of the above nef gene to CAT and newly inserting an Nde I site
19 ・ (Nde) was prepared. Nef19 ・ (Nde) to Nde I and Hi
The NdeI-HindIII fragment of about 0.82 kb was cut with ndIII, and the resulting expression vector pT7-cleaved with NdeI and HindIII.
No. 7 was inserted and ligated to prepare pT7-Nef. pT7-Nef to BL
When introduced into the 21 (DE3) strain, more than 10% of the total protein was expressed and accumulated in Nef protein.

Example 19 Extraction and Purification of Nef Protein: Transformant Clone BL21 (DE
3) 37 / pT7-Nef in LB medium containing 20 μg / ml ampicillin
Cultured at 18 ° C for 18 hours. To a fresh LB medium (containing 20 μg / ml ampicillin), add the above culture solution to 1/100 volume equivalent, and add
The cells were cultured at ℃. When the turbidity OD _{600nm of the} medium reaches 1.0,
IPTG was added to 1 mM, and the cells were further cultured at 37 ° C. for 5 hours. Collect cells by centrifugation (5,000 rpm, 10 minutes),
Suspended in 1/50 volume of the culture solution, 20 mM sodium phosphate pH 7.0, and sonicated (30 times treatment 6 times, 19.5 kHz, 300 W)
The supernatant obtained by centrifugation (19,000 rpm, 60 minutes) was separated as a crude extract.

Rough purification of Nef protein: Ammonium sulfate was added to the crude extract to 35% saturation, and the mixture was stirred and centrifuged (16,000 rpm,
20 minutes). The resulting precipitate was washed with 20 mM tetrium phosphate.
It was dissolved in pH 7.0 and dialyzed with the same solution.

Purification of Nef protein: The dialyzed sample was passed through S-sepharose (Pharmacia [Sweden]) equilibrated with 20 mM sodium phosphate pH 7.0. The fraction containing the Nef protein was eluted with a 0 to 1 M sodium chloride gradient, and dialyzed against 20 mM sodium phosphate pH 7.0. this,
The mixture was passed through a hydroxyapatite column (KB column, manufactured by Koken Co., Ltd.) and eluted with a 20 to 700 mM sodium phosphate gradient. The fractions containing the Nef protein were pooled and dialyzed against 20 mM Tris-HCl pH 8.3, 5 mM 2-mercaptoethanol. The sample was passed through a MonoQ column and eluted with a 0-1 M sodium chloride gradient. The fractions containing the Nef protein were pooled and used as purified Nef protein. About 7 mg of purified Nef protein could be obtained from one transformant culture.

The Nef protein produced in large quantities in E. coli and purified had the N-terminal methionine residue removed, but was not myristoylated, and the N-terminal amino acid sequence was Gly-Gly-Lys-T
rp-Ser-Lys-Ser-Ser-Val-Ile-Gly-Trp-Pro-A
la-Val -... coincided with the amino acid sequence predicted from the nucleotide sequence.

Example 20 Diagnosis of HIV-1 Using Purified Nef Protein: The purified Nef protein prepared in Example 19 was electrophoresed on polyacrylamide as described in Example 2, and then blotted on a nitrocellulose membrane. Later, for blocking, the membrane was
% (W / W) gelatin solution. The presence of antibodies to HIV-1 Nef protein was determined by human
The test was performed using the serum of an HIV-1 carrier (7 cases).
Serum from healthy adults (nine cases) was similarly tested.
Table 2 shows the results.

Six of the seven HIV-1 carriers reacted positively with the Nef protein and were positive. These results indicate that the use of the purified HIV-1 Nef protein produced and prepared in Escherichia coli according to the present invention enables specific detection and diagnosis of HIV-1 infection.

Effects and Effects of the Invention (1) According to the present invention, retroviral gene large-scale expression and processing can be simultaneously performed, and retroviral antigens and enzymes using the expression vector are extremely dangerous. Since the virus itself is not used, it is excellent and safe from the viewpoint of biohazard countermeasures during the manufacturing process, and the manufacturing operation is easy.

(2) The production amount of each core protein, each enzyme, and Nef protein in the culture solution 1 is 1 to 30 mg in terms of protein amount, and the production yield is extremely high.

(3) According to the present invention, each core protein of the retrovirus,
Although protease, reverse transcriptase, and integrase are expressed as fusion proteins with a gene product that is overexpressed, they can be produced as processed single mature proteins instead of fusion proteins. It is more efficient and rational than the expression of each of the above-mentioned genes alone, and when the above-mentioned (1) and (2) are also taken into account, the production cost is low and economical.

(4) Since an enzyme having extremely high specificity for a substrate specific to a retrovirus and various antigens of the virus with high purity can be provided at a low cost and in a large amount, not only genetic engineering but also retrovirus infections such as AIDS, In addition to the fundamental research on adult T-cell leukemia, chicken sarcoma and leukemia, feline leukemia, etc., the development of specific therapeutic and prophylactic agents with a high degree of selective toxicity, diagnosis, etc. It is the gospel for the promotion of livestock.

(5) The method for preparing a plasmid according to the present invention enables the expression of various genes of a retrovirus and the various genes of a retrotransposon in large amounts and subsequent processing. It can be applied to the development of efficient mass production.

[Brief description of the drawings]

FIG. 1 shows the reverse transcription activities of Escherichia coli transformed with the plasmid pPG280 having the HIV pol gene and a crude extract of Escherichia coli transformed with the vector pUR29 having no HIV pol gene. Fig. 2. Plasmid pPG28 having the HIV pol gene and HIV
5 shows the results of Western blot analysis of crude extracts of Escherichia coli each transformed with the vector pUR290 having no pol gene using HIV carrier serum. FIG. 3 shows an elution pattern of reverse transcriptase of a crude extract of E. coli by anion exchange column chromatography. FIG. 4 shows the separation of reverse transcriptase by Affigel heparin chromatography.

Continuation of the front page (72) Inventor Atsushi Nakata 6-24-1-201 Higashi Toyonaka-cho, Toyonaka-shi, Osaka (56) Reference Proc. Natl. Acad. Sc i. USA, Vol. 84, p. 8903-8906 (1987) Science, Vol. 231, p. 1580-1584 (1986) Proc. Natl. Acad. Sc i. USA, Vol. 86, p. 9549-9553 (1989) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 C12P 21/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A plasmid that expresses the HIV gene in Escherichia coli UT481, wherein the plasmid lacZ of the plasmid pUR290 series is used.
The HIV gene cDNA fragment is inserted and linked in the same frame within the gene, and the inserted cDNA fragment is linked to the next HIV gene cDNA from left to right in the direction from the 5 'end to the 3' end.
P17, p24 and protease; p24, p15 and protease; p24, p15 and protease; p17 and protease; p24 and protease; and p15 And proteases. 2. A plasmid that expresses the HIV gene in Escherichia coli UT481, wherein the plasmid lacZ of the plasmid pUR290 series is used.
It is created by inserting and linking the HIV gene cDNA fragment in frame with the frame, and
The NA fragment is a plasmid selected from the following HIV gene cDNA fragments, described from left to right in the direction from the 5 'end to the 3' end: a plasmid: p17, p24, p15 and protease; p17 P24, p15 and protease; p17, protease; p24 and protease; and p15 and protease. 3. A plasmid that expresses the HIV gene in Escherichia coli BL21 (DE3), wherein an HIV gene cDNA fragment is inserted and linked in the vicinity of the downstream of the promoter of plasmid pT7-7. A method for preparing a plasmid, which is one fragment selected from the following HIV gene cDNA fragments, described from left to right in the direction from the 3 'end to the 3' end: Protease; Protease and reverse transcriptase; Reverse transcriptase Proteases, reverse transcriptases and endonucleases; p17, p24, p15 and protease; p17, p24 and protease; p24, p15 and protease; p17 and protease; p24 and protease; and p15 and protease. 4. A plasmid expressing the HIV gene in Escherichia coli BL21 (DE3), which is produced by inserting and linking an HIV gene cDNA fragment in the vicinity of the downstream of the promoter of plasmid pT7-7, and The fragment is one fragment selected from the following group of HIV gene cDNA fragments, described from left to right in the direction from the 5 'end to the 3' end: Protease; Protease and reverse transcriptase; Reverse transcription Proteases and reverse transcriptase and endonuclease; p17, p24, p15 and protease; p17, p24 and protease; p24, p15 and protease; p17 and protease; p24 and protease; and p15 and protease .