JPS63503356A - Expression of human T-cell lymphotropic virus (HTLV-3) reverse transcriptase and its use - 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 14. A bacterial host cell according to claim 13. 15. The yeast host cell according to claim 13. 16. A bacterial host cell comprising the 1 lasmid of claim 1. 17, as set forth in claim 13, deposited under accession number ΔTCCNo. 67117. E. coli strain 11 BIOI. 18. The host according to claim 13 under suitable conditions capable of producing the polypeptide. 1. A method for producing a polypeptide having HTLV-I reverse transcriptase activity, which comprises growing main cells and collecting the obtained polypeptide. 19. The host according to claim 16 under suitable conditions capable of producing the polypeptide. 1. A method for producing a polypeptide having HTLV-[) reverse transcriptase activity, which comprises growing main cells and collecting the obtained polypeptide. 20. The host according to claim 17 under suitable conditions capable of producing the polypeptide. 1. A method for producing a polypeptide having HTLV-III reverse transcriptase activity, which comprises growing main cells and collecting the obtained polypeptide. 21. A polypeptide having 11TLV-1 reverse transcriptase activity prepared by the method according to claim 20. 22. Plasmid p) A polypeptide having reverse transcriptase activity, characterized in that it is encoded by IRT22. 23. HTLV-III reverse transcriptase was isolated and an appropriate amount of deoxyribonucleate contacting the isolated reverse transcriptase with the RNA molecule to be reverse transcribed in the presence of a compound mixture to produce a transcription mixture; and contacting the transcription mixture with an amount of the substance to be identified. to detect the amount of reverse transcriptase activity in the mixture and detect the amount of reverse transcriptase activity in the mixture that does not come into contact with the substance. 1. A method for identifying a substance that inhibits HTLV-I reverse transcriptase activity, characterized in that a significant decrease in the amount of reverse transcriptase activity relative to the amount of reverse transcriptase activity is used as an indicator of inhibition of reverse transcriptase activity. 24, physiologically acceptable HTLV-reverse transcriptase according to claim 23 1. A method for treating a patient with a disease associated with the HTLV-11i virus, which comprises administering to the patient an appropriate amount of a primary activity inhibitor for an appropriate period of time. Specification: Human T-cell lymphotropic virus (HTLV-111-7, Nobino Fatbite 1) The present invention was developed by the National Cancer Institute of the United States Department of Health and Human Services. Grant from itute) Number CA 30488 was made with support from the government. The United States Government owns a portion of the rights to this invention. In the specification of the present application, reference is made to several documents, the reference numbers of which are given in parentheses. A complete list of these documents is attached at the end of this specification, immediately preceding the ``Claims.'' In order to more clearly indicate the current state of the technical field of the present invention, this specification is based on these documents. The entire disclosure in this publication is incorporated by reference. Acquired immunodeficiency syndrome (AIDS) is a disease characterized by a severe lack of cellular immune response. It is a new epidemic disease with unique characteristics. The substance that causes this disease is now known as human It is believed to be a human retrovirus known as human T-cell lymphotropic virus III or lymphadenopathy virus (HTLV-III/L^) (1-4). In order to suppress the spread of this virus, one of the keystones is to give individuals resistance to infection by immunizing them. The other is the development of antiviral vaccines that can The aim is to develop antiviral drugs that can specifically slow or stop the production of viruses. drugs like this One potentially important target for the drug is the pyrion-binding enzyme reverse transcriptase. (5-8). In the early stages of the retrovirus life cycle, the viral RN^ is copied to form double-stranded DNA, and this double-stranded DNA is integrated into the host DNA to generate a provirus (1). Synthesis of proviral DNA involves an enzyme called reverse transcriptase. be a medium. This synthesis can effectively utilize an RNA or DNA template to perform DNA synthesis by extension of primers carrying a pair of 3' hydroxyl termini. This protein contains a second activity, RNA5e H, which is responsible for the complementary RN^:DN^ pol gene, which is involved in multiple activities involved in various steps of the life cycle. Encodes many enzyme activities. The pol gene product was initially produced by a pol gene product, which is a fusion of the sequence encoded by the pol gene and the gag gene. Protein Pr200gag-po! (2,3). Proteolytic treatment is required to remove the gag sequence and excise the mature product from the pol sequence. Reverse transcriptase is widely used as a means to produce complementary DNA (cDN^) copies of the Metsenger RN^ (+RN^) molecule. These cDN^ can be inserted into expression vectors used to transform cells and cause the resulting cells to produce the desired polypeptide encoded by the original mRN^. +1TLV-111 reverse transcriptase has been purified in small quantities and some of its properties have been established. Since this enzyme has a rare template and prefers divalent cations (9-11), it is possible that specific inhibitors of this enzyme will be discovered. A class of inhibitors of HTLViI reverse transcriptase activity can be used to suppress the activity of the enzyme when it is produced by a virus that infects human cells and tissues. Since reverse transcriptase is an essential part of the viral life cycle, the use of such inhibitors may be used to treat or prevent HTLV-III related diseases. It seems to be that. Isolation of such inhibitors may be difficult if the active enzyme is available in large quantities. It will be easier if you do. Reverse transcriptase produced by and isolated from pyrions is commercially available. However, due to the small amount of the pol gene product in pyrions, commercially available reverse transcriptase is quite expensive. Some of the techniques already disclosed in this field are based on genetically engineered vectors. It involves the production of polypeptides with reverse transcriptase activity by bacteria transformed with -. One of these prior art methods uses shotgun cloning into Escherichia coli of complete genomic DNA isolated from vertebrate cells, such as poultry hepatocytes. (Japanese Patent Publication No. 56087600). Two other prior art methods have been developed to produce high yields of reverse transcriptase in E. coli. Rovirus po! Discloses the expression of segments of genes (16.17). No. 731,128, filed on May 6, 1958 and co-pending, discloses an enzymatic reversal of activity produced in relatively high yield from Mooney murine leukemia virus (MuLV). The expression of photoenzymes has been described. The present invention provides HTLV-I11po! inserted into a plasmid! Use modified regions of genes. Its transcription is regulated by an inducible promoter. This inserted gene file Expression of the fragment produces a polypeptide with HTLV-III reverse transcriptase activity. The invention also relates to a method for identifying substances that inhibit HTLV-III reverse transcriptase activity. The present invention relates to a double-stranded DNA plasmid which, when expressed in a suitable host cell, produces a polypeptide having 1 (TLViII) reverse transcriptase activity. - a DNA sequence containing an inducible promoter and a -^TG opener. A DNA sequence containing an initiation codon and -encoding a polypeptide with reverse transcriptase activity. - a selectable or identifiable phenotype expressed when the vector is present in a host cell; - a DNA sequence containing a gene associated with the property; and - a DNA sequence containing an origin of replication from a bacterial plasmid capable of autonomous replication within the host cell. column contains. The plasmid of the invention can be introduced into a suitable host cell in which the gene can be expressed under suitable conditions. In a currently preferred embodiment, the plasmid is pHRT25 and the host cell is E. coli HBIO1 cells (both donated under TCC No. 67117). (entrusted). Suitable inducible promoters are promoters that are induced when host cells are grown in a medium lacking one or more amino acids. One such inducible promoter is present in the Trp operon of E. coli. The invention further relates to a method for identifying substances that inhibit HTLV-111 reverse transcriptase. Ru. This method involves isolating HTLV-III reverse transcriptase and contacting the reverse transcriptase with the RN conjugate to be reverse transcribed in the presence of an appropriate amount of deoxyribonucleotides. Reverse transcriptase activity in the cocktail can be detected by preparing a transcription cocktail and contacting the transcription cocktail with an amount of the substance to be identified for a suitable period of time. Therefore, the amount of reverse transcriptase activity in the cocktail that has not been brought into contact with the substance is If the amount is significantly decreased compared to the sexual amount, it means that the reverse transcriptase activity is inhibited. The present invention also relates to methods of treating diseases associated with the HTLV-III virus. The method comprises administering to the patient a suitable amount of a physiologically acceptable HTLV-111 reverse transcriptase inhibitor over a suitable period of time. Part (7), "7'; B Figure 1 ' Configuration of 1) 3.7kb flag to ITLV-1flllN (provirus that was cloned) The protein was excised by cutting with Bgl II + Sal I and inserted into the expression plasmid at the Ba...H1 and Sal I sites. Cloning operations are prior art Therefore, it was carried out (16-18). Similarly, the same pol sequence was inserted in the wrong reading frame into the pAT)13 vector to form plasmids pHRT31 and p)lRT32. Figure 2 HTLV-III 01. This map shows the restriction site within the vector (pAT) 12 (dark line) and the restriction site within the insert (pol region of IITLV-IN, light line). Based on the HTLV-111 map of Ratner et al., the insertion start point is near nucleotide 1640 (I (Bgl11 site of TLV-111)) and the insertion end point is near nucleotide 5368 (5alI site of HTLV-III). (12). Cultures were grown in the absence of tryptophan and lysed according to the prior art (14-17), Panel A: shown here. (carrying a plasmid) IBIOI cells. Each extract 1.1 or 0.1.1 was incubated in a reaction cocktail of 5opI containing 50mM of pl(8,3) Tris-flcl and 30mM of -DTT, and leotide (ICi/mmol). .The reaction is shown on the left. Sea urchin contained Mg++ (10d) or Mru+ (1mM). The column labeled ΔT contains the substrate poly(r^) (101 g/ml) and oligo(dT) (hg/ml), and the column labeled GC contains the substrate poly(rC) (1000 g/ml). ml) and oligo(dG) (hg/ml) both contained Col. The reaction was allowed to occur for 30 min and 10-1 was spotted onto DEAE paper (DE81 Jhatman). It was stopped by hitting the button. The paper was washed in 2x SSC (0.3M NaCl, 0.03M citric acid +) for 3x10 minutes each at room temperature, rinsed twice with 95% ethanol, dried, and transferred to X-ray film. exposed to light Ta. Samples numbered and C are assays of Mooney murine leukemia virus-prone authentic reverse transcriptase and control cell growth medium, respectively. Panel B: HBIOI (PolA+) and C2110 (Pal^l) cells carrying the plasmids shown here were used. Assay and substrates are the same as for panel A. When expressed in a suitable host, a double-stranded DNA plasmid was formed that produced a polypeptide with TLV-III reverse transcriptase activity. ITLV-111po1 (containing a DNA sequence containing an inducible promoter, a DNA sequence containing an ATGT start codon, and a DNA sequence encoding a polypeptide with human T-cell lymphotropic virus III (HTLV-III) reverse transcriptase activity) A genetic moiety and a selectable or identifiable phenotypic characteristic, e.g. A DNA sequence containing a gene associated with drug resistance, such as ampicillin resistance, expressed when the target is present in a host cell, e.g. l) N^ sequence containing an origin of replication from a bacterial plasmid that can autonomously replicate within cells; nothing. In one embodiment of the invention, the inducible promoter of the plasmid is a promoter that is induced when host cells are grown in a medium lacking one or more amino acids. Such a promoter may be, for example, the E. coli dirρ promoter, and the deleted amino acid in the medium may be tryptophan. In another specific example, an induced prism The promoter is a promoter that is induced when host cells are exposed to higher temperatures. The ^TGT start codon of the plasmid is the codein of the E. coli TrpE protein. for example, a sequence of approximately 1000 nucleotides encoding a portion of the E. coli TrpE protein. - In a specific example, the origin of replication is derived from pBR322. The plasmid has the restriction map shown in Figure 2 and was donated under TCC No. 67117. As for the plasmid identified as pHRT25 in the entrusted E. coli 11 BIOI. It can be a circular double MDNA sequence. The HTLV-III pol gene portion of the plasmid may contain a nucleotide sequence from about nucleotide 1640 to about nucleotide 5368. -Specific examples and Thus, the 5' end of the central portion of the pol gene is located 21 nucleotides from the starting point of the DNA sequence encoding a polypeptide with reverse transcriptase activity. Produce a DNA vector and transform appropriate cells to obtain reverse transcriptase activity. The methods used to produce these polypeptides are known to those skilled in the art and are described in more detail in the detailed experimental description below. Traditional cloning vehicles such as plasmids, e.g. pBR322, are used to clone new cloning vehicles containing DNA encoding non-naturally occurring polypeptides with reverse transcriptase activity. To obtain new cloning vehicles, modifications can be made using known methods, but other methods can also be created. Similarly, such loning vehicles can be modified or created to contain ON^ sequences, i.e., inducible promoters (such as the Trρ promoter), which are involved in the regulation or expression of sequences encoding polypeptides with reverse transcriptase activity. can be done. The ON heptagonists inserted in this manner can be formed by a variety of methods, including enzymatic or chemical synthesis. The cloning vehicle thus obtained is a chemical that does not occur in nature and can only be produced by modern technology, commonly referred to as recombinant DNA technology. These cloning vehicles can be prepared using methods known to those skilled in the art, such as transformation, I. They can be introduced into suitable host cells, such as prokaryotic cells such as bacteria (such as Escherichia coli or Bacillus subtilis) or eukaryotic cells such as yeast, by transfection or the like. One specific example of the present invention is Escherichia coli 1 (8101 strain) containing the plasmid pi (RT25) deposited under TCC No. 67117. In that case, the cells into which the plasmid of the present invention is inserted have reverse transcriptase activity. The expression of the DNA encoding the non-naturally occurring polypeptide is regulated by the Trp promoter.As a result, reverse transcriptase enzyme #il cells were obtained into which DNA encoding a non-natural polypeptide encoding activity and the Trp promoter was introduced. polypeptide with The cells may be grown under suitable conditions known to those skilled in the art to produce the polypeptide and recover the resulting polypeptide. One embodiment of the invention therefore relates to a polypeptide prepared as described above, for example a polypeptide with reverse transcriptase activity characterized in that it is encoded by plasmid pHRT25. Another embodiment of the invention is a method for identifying substances that inhibit HTLV-1 reverse transcriptase. The method involves isolating HTLV-1 reverse transcriptase and combining the isolated reverse transcriptase with the RN protein to be reverse transcribed in the presence of an appropriate amount of deoxyribonucleotides to produce a transcription cocktail. The transcription mixture is contacted with an amount of the substance to be identified for an appropriate period of time, the amount of reverse transcriptase activity in the mixture is detected, and the amount of reverse transcriptase activity is compared to the amount of activity in the mixture without contact with the substance. A significant decrease in the amount of enzyme activity is an indicator of reverse transcriptase activity inhibition. In a preferred embodiment, HTLV-ill reverse transcriptase will be isolated from a cell, such as a bacterial or yeast cell containing a plasmid containing the HTl, V-11pol gene, such as plasmid pHRT22 or pHRT25. HTLV-III reverse transcriptase can also be used to infect other sources, such as the IITLV-111 virus. can be isolated from stained tissue culture cells. 1 (used in the production of V-I reverse transcriptase) Methods for performing such experiments are known in the art and are described more fully below in the detailed experimental description. This invention should not be used! -'i = The silibonucleotide mixture will contain dATPl, :I (:TP, dC'A) and dTTi) in relative amounts appropriate for each experiment. In a preferred embodiment, reverse transcriptase is used to detect the amount of reverse transcriptase activity. one or more radioactively labeled deoxyribonucleotides, e.g. For example, the amount of 32p-^TP is detected by ol-radiography. another person Methods such as the use of quantitative immunoassays are also possible. According to yet another embodiment of the invention, there is provided a method of treating a patient with a disease associated with the IITLV-111 virus. An appropriate amount of the transcriptase activity inhibitor is administered to the patient for an appropriate period of time. The inhibitor may be administered in a variety of ways, including as an injectable solution in a pharmaceutically acceptable carrier. For example, they may be administered intravenously or intraperitoneally, or may be applied topically or orally in liquid or solid form, or by a variety of other routes known in the art. I can give. HTLV-1 [To facilitate the preparation of the enzyme, a gene fusion was prepared between the E. coli trpE gene and an appropriate portion of the viral pol gene. pol's remains A large DNA fragment (containing almost the entire gene) is converted into a biologically active protein. virus clone (pHXBc2; (12)) and fused the 5' half of the trpE gene to the above pol sequence in the correct reading frame to create a ρA T 112 expression vector. Insert the two overlapping clones of the resulting plasmid into the vector (Figure 1). Select pHRT22 and pHRT25 for analysis. Figure 2 shows one black Figure 2 shows a detailed restriction map of pHRT25. Clones pHRT22 and p)I RT25 have essentially similar restriction maps. A similar procedure was used to insert the pATH3 vector in open reading frame with the same pot sequence to form control plasmids pHRT31 and pHRT32. Bacterial cultures containing various 1 lasmids were grown in minimal medium lacking trigtophan and contacted with indoleacrylic acid for derepression of the TRP operon (14-17); after 2 h, cells were harvested and Prepare a lysate and test the reverse transcriptase activity of the crude extract. The properties of the two substrates were directly assayed using the method described above (16-18) (Fig. 3). Poly(r^) or oligo(dG) with oligo(dT) as primer Add appropriate radioactive triphosphate to each poly(rC) to be used as an imer. The ability of the extract to absorb Each substrate was tested with two divalent cations: Hg2'' (IOB) or Mn'' (1 mM). All cultures, regardless of the type of plasmid they carried, showed significant background activity on the poly(rΔ):oligo(dT) substrate. Slightly increased levels of activity were detected in cells harboring the constructed pHRT22 and pHRT25. In contrast, poly(rC):oligo(dG) substrates showed dramatic results. vector -Cells harboring pHRT22 and pHRT25 showed virtually no background activity, whereas cells harboring pHRT25 showed high levels of activity (Figure 3, p. Nel A, 2nd row). The activity was highly specific for the divalent cation Mg24 (compare rows 2 and 4). This trend is markedly different from murine reverse transcriptase activity. Mouse reverse transcriptase activity is highly active on poly(Δ):oligo(clT) templates in the presence of Mn2+ (column ■). A control plasmid formed by fusion of the wrong open reading frame shows only a trace amount of activity just above background. Background of DNA polymerase activity for poly(r^), oligo(dT) type I Plasmids were added to bacterial strains lacking DNA polymerase I to reduce rounding. (18), these cells do not support replication into plasmid DNA. Thus, transformants carry plasmid DNA that has been integrated into the host chromosome in one or only a few copies. Extracts were prepared and assayed as described above (Figure 3B). Pol^- carrying the vector plasmid pATl12 can contain any divalent cation. showed no detectable background activity for either template in the presence of I didn't. These same cells harboring the constructed pHRT22 showed high levels of activity in the presence of Mg2+. Activity could be detected for both poly(rC):oligo(dG) and poly(r^):oligo(dT), but slightly higher activity was shown for the poly(rC):oligo(dfl:) template. . In the presence of Mn2+, almost no activity was detected for both templates. These properties of activity are exactly the same as the behavior of authentic HTL V-I reverse transcriptase produced from pyrion particles (9-11). To quantitatively measure the activity level, poly(rC):o in the presence of H,2+ Repeat the assay on Rigo(dG) template and scintillate the amount of radioactivity incorporated. Measured by ration counting. Assays with missing primers or templates data are not shown to show only background activity). Titration of the extract during the 10 minute reaction showed that the assay was linear up to 1 μp of extract. (Table I). Extracts of cells harboring the pHRT22 construct showed a 200-fold increase in specific activity compared to extracts from control cells. Analyzing the reaction over time using 0.1 μl of extract indicates that the reaction does not occur directly for more than 10 minutes. These experiments also showed that the cells harboring the constructed pHRT22 had significantly increased specific activity compared to the control, which was found to be linear (Table ■). The specific activity of Po1A- cells containing pHRT22 was approximately 175 times that of Po1 A+ cells harboring the same plasmid. The reason for this is that positive It is presumed that this is due to the small number of copies of mido. In conclusion, we demonstrated that the HTLV-IItpol gene was inserted into a bacterial expression plasmid and that this plasmid induced reverse transcriptase activity. The appearance of activity depends on whether the trpE and pol sequences are joined in the correct reading frame, and the resulting activity is similar to the bacterial polA gene, with M,2+ being preferred over Mn2+. The activity differs markedly from that of the enzyme encoded by murine leukemia virus. These constructs and more active derivatives will be useful in the genetic analysis of the function of the ITLV-IU pol gene, and will also be useful in the investigation of antiviral agents. Titration of Extract Amount of Extract Uptake clGTP Specific Activity HBIOI (pATH2) OO o,1 <0.035 ≤0.02 HBIOI (pltRT22) OO Procedure Extracts from the indicated cultures were prepared as described above. 16-17). The indicated amounts of the extract were combined with 50mM TO1CN pl+8.3, 20+nM DTT, 60IQM NaCM', 0.05% NP40 and 10mM MI? A reaction mixture containing Cf f2, 10 IHt/x1 poly(rC), 5 μg/IR oligo(dG), and io μs of '2P-dGTP (ICi/mmole) was incubated in a total volume of 50 μl. The reaction mixture was incubated for 10 minutes at 37°C and spotted on a grid of DEAE paper, the paper was batch washed with 2x SSC, rinsed with ethanol and dried, and the number of incorporated counts was determined by scintillation counting. Nucleotide uptake i A background count of 220 CPM, which was present in the reaction without extract, was subtracted before calculating the (pmole). Protein concentration quantification using the method of Braclford (19) revealed that the concentration of HBIOI (p AT) 12) was 44 CPM. /Takip, the concentration of HBIOl, (pHRT22) was 3.3 wgZi.The activity of the extract was calculated from the slope of the least squares fit of the titration data.Table ■ Time course of uptake Cells Strain Time Uptake dcTP Specific Activity (min) (pmoffi/μg (pII I017μg protein) Bacterium 710 min) HBl, 01 (p to TH2) O0 HBIOI (pHRT22) O0 Cello (pATH2) OO An <0-07 C2110 ( ptlRT22) 0 01 0.004 2 0.06 0.30±0.03 Procedure 0.1μ! of crude extract of the indicated culture was incubated in the reaction mixture for the indicated time. The number of counts captured was determined as in Table I. Backup before activity calculation Ground activity 150-200 CPM was subtracted. The protein concentration was as follows. HBlol (pATH2): 4.4zg/zN; HBIOI (pH RT22): 3.3zy/, vZ+C2110 (pATf12): 3.9 shoulder g/z1); C2110 (pHRT22): 5.2xg/x1. Specific activity was measured as described above. Hakubamumomi 1. R, C, Ga1lo et al., 22Q, p 865 (1983). 2.! ″, Barre-5 inoussi-et al-y spoon-2≧22 L 868 (198]), - 3, R, C, Ga1lo et al-z danΩ1 Ωa22it 500 (1984)--4, -A, S, Fauci et al, er ed 3 J) fJ-, 92 (1984). 5, P, Chandra, r!at991974). 6, E, DeClercqr F JL/ 9 (1979). 8, H, Mitsuya et al, I SΩ1MiyuΩdan22B-1172 (1984). 9@M, A, Rey, B-5pire, D, Dormont, F'= Earl e-5inoussi, L, Montagnier, and: J, C, Che r+nann.”' V I 12 Yu, 126 (1ri84). 10, P., Chandra, A., Voge and T-Gerber, C≧Tech; 11. A, D, Hoffrr+an, B, Banapour, and J, A,, LeVy,. yL theory, Eshimi, 326 (1985). 12, Patner et al., 277 (1985). 13 T. J. Koerner, Duke University; unpublished. 15, D.G., Kleidetal, C.Lx., 1125 (1981). 17, M, Flo Shichiri, N, Tanese, and S, P, Gaff, Mu J 22 Ω2 = 1 small LO-, 9326 (19851°18, S, P, Goff, P, Trakt:nan, and D , Balse 1 moret lxy 辷IE, present L 2] 9 (19811゜19, M, M, Bradford, '-' Lazy 瑳工に u, 248 (1976). FIG+JR1!1 F!+1; IJRE 2 FIGURE 3 International search report PCT/US37101162 CLASS engineering F engineering CAT engineering ON OF 5UBJECT MAT”, ER (CONT engineering NUED) any F engineering ELDg 5EARCHED (CONT engineering NU ED)

Claims (24)

[Claims] 1. When expressed in a suitable host cell, a protein with HTLV-III reverse transcriptase activity is produced. DNA sequence containing an inducible promoter in the 5' to 3' direction. sequence, a DNA sequence containing the ATC start codon, and a polypeptide with reverse transcriptase activity. Human T-cell lymphotropic virus (HTLV- III) substantially all of the pol genes; A selectable or identifiable expression expressed when the vector is present in a host cell a DNA sequence containing a gene associated with type characteristics; a DNA sequence containing an origin of replication from a bacterial plasmid that can autonomously replicate within a host cell; A double-stranded DNA plasmid containing. 2. Claim 1, characterized in that the suitable host cell is E. coli. Plasmid. 3. An inducible promoter drives host cells in a medium lacking one or more amino acids. Claim 1, characterized in that it is a bromotor that is induced upon proliferation. Plasmids described in. 4. The inducible promoter is the E. coli Trp promoter, and the medium is tryptopophilic. 4. The plasmid according to claim 3, which is a fan-deficient medium. 5. The ATG start codon is derived from the coding sequence of the E. coli TrpE protein. The plasmid according to claim 4, characterized in that: 6. The ATG start codon encodes a portion of the E. coli TrpE protein. Claim 5, characterized in that it is derived from a sequence with 000 nucleotides. Plasmids described in Section. 7. Inducible promoters are promoters that are induced when host cells are exposed to high temperatures. The plasmid according to claim 1, which is a plasmid. 8. The protein according to claim 1, characterized in that the phenotypic characteristic is drug resistance. Lasmid. 9. Claim 1, wherein the origin of replication is derived from pBR322. plasmid. 10. The protein according to claim 1, wherein the double-stranded DNA is circular. Lasmid. 11. Identified as pHRT25 with the restriction map shown in Figure 2 and accession number ATC. CNo. No. 67117 in E. coli HB101 as set forth in claim 1 plasmid. 12. HTLV-IIIpo1 residue from about nucleotides 1640 to about 5368 The plasmid according to claim 1, which contains substantially all of the gene. 13. A suitable host cell containing a plasmid according to claim 1. 14. A bacterial host cell according to claim 13. 15. Yeast host cell according to claim 13. 16. A bacterial host cell comprising a plasmid according to claim 1. 17. Accession number ATCC No. Claim 13 filed under No. 67117 E. coli HB101 strain. 18. The host according to claim 13 under suitable conditions capable of producing the polypeptide. HTV- characterized by proliferating main cells and collecting the obtained polypeptide. Method for producing a polypeptide having III reverse transcriptase activity. 19. The host according to claim 16 under suitable conditions capable of producing the polypeptide. HTLV characterized by proliferating main cells and recovering the obtained polypeptide -III A method for producing a polypeptide having reverse transcriptase activity. 20. The host according to claim 17 under suitable conditions capable of producing the polypeptide. HTLV characterized by proliferating main cells and recovering the obtained polypeptide -III A method for producing a polypeptide having reverse transcriptase activity. 21. HTLV-III reverse prepared by the method of claim 20 A polypeptide with transcriptase activity. 22. Reverse transcription enzyme encoded by plasmid pHRT22 A polypeptide with elementary activity. 23. HTLV-III reverse transcriptase was isolated and an appropriate amount of deoxyribonucleotide Connecting the isolated reverse transcriptase and the RNA molecule to be reverse transcribed in the presence of the mixture. contacting to produce a transfer mixture; and contacting the transfer mixture with an amount of the substance to be identified. , detecting the amount of reverse transcriptase activity in the mixture, and detecting the amount of reverse transcriptase activity in the mixture that does not come into contact with the substance. A significant decrease in the amount of reverse transcriptase activity relative to the amount of A method for identifying an HTLV-III reverse transcriptase activity inhibitor, characterized by: 24. Physiologically acceptable HTLV-III reversal according to claim 23 HTL characterized in that an appropriate amount of a transcriptase activity inhibitor is administered to a patient for an appropriate period of time. A method for treating patients with V-III virus-related diseases.