JPS63502000A - AIDS virus gene expression - 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] AIDS virus gene expression field of invention The present invention relates to the field of molecular biology, and more particularly to the field of molecular biology, and more particularly to Concerning the expression of genes from II viruses and their use.

Background of the invention Human T lymphotropic virus type-III (HTLV-III) AIDS virus (LAV) or AIDS-associated retrovirus (ARV) It is also known for its role in acquired immunodeficiency syndrome (AIDS) and related diseases. It is a pathogen. HTLV-III is evolutionarily more closely related to ungulate lenticules. It is related to the human T-lymphophilic virus that was previously isolated. type I and II (HTLV-I and HTLV-II) and many common characteristics. especially those that infect helper 'l'-IJ lymphocytes and impair immune function. share their biological and pathogenic properties that are a consequence of their abilities. Sara In addition, certain exceptional characteristic units HTLV types IS ■ and ■ are associated with bovine leukemia. Viruses and simian T-lymphophilic virus type I and ungulate leprosy Animal retroviruses such as chi-retroviruses, i.e. virus long ・Virus that mediates transcriptional activation initiated at terminal repeats (LTR) It is related to the presence of coded proteins. This transcription activator (tat ) are critical for the biological activity (transformation or cytopathicity) of this group of viruses. It is assumed that this plays a role.

The severity of AIDS depends on early and accurate diagnosis of infection with HTLV-III and makes the detection and elimination of HTLV-■ contaminated samples from blood banks extremely important. do. Ga1lo et al., U.S. Pat. No. 4,520,113 , to HTLV-III to detect the presence of anti-HTLV-nu antibodies in serum. discloses the use of the derived antigens. Montagnier et al. et al.), European Patent Application No. 138667, describes the Discloses the use of specific HTLV-I antigens to detect infection. Papa Papas, et al., U.S. Patent Application No. 6-664,972 (Der. Derwent Accession No. 85- 110268/18) is E--:l! 17) HTI, V-l-1-embe Detecting expression of rope protein coding sequences and infection by HTLV-I Discloses the use of the protein thereby expressed for. Crawl “ Jill (Crowl et al, Ce1l), 41,979 (to 198 □ 5) is a part of the HTLV-I [1 envelope protein gene env]. Such a protein for expression in E. coli and detection of infection by HTLV-III. reported using proteinaceous substances. Kasei et al., Journal of Pyrology (C a5ey et al., J. Virol, ), 55.417 (1985) purification of the gag gene product, an internal structural protein of HTLV-III called p24. protein and the use of the protein to detect infection by HTLV-■. Reporting.

Seiki et al., Proceedings of the National Academy of Sciences Yences of Knee Nice Knee (5eiki et al, Pro c, Nat'l, Acad.

Haseltine et al., 5science ), 225.419 (1984) are tat-1 and HTLV-II, respectively. report the identification of a protein that mediates the transcriptional activation of LTR in .

Sodroski et al., Science.

In trans state of gene expression from LTR in ■-■ ) reports activation.

Arya et al., Science (Arya et al., 5science), 2 29.69 (1985) and Sodroski et al., Science (5odroski et al, 5science) %22'9.74 (1985) is tat- The tat protein encoded by HTLV-I [1, designated as HTLV-I [3] and the t Identification and cloning of cDNA encoding at protein in E. coli reporting.

Summary of the invention In one embodiment, the present invention provides a DNA coding system operably linked to a regulatory element. The DNA coding sequence consists of the HTLV-I [I tat-3 encodes a protein or a derivative thereof, and the derivative is Potatoes that are reactive with antiserum against tat-3 elicit a response to infection. -Relates to an E. coli expression vector characterized by being a repeptide.

In another embodiment, the invention provides a method for combining a sample of serum from an animal with tat-3 or has been shown to induce a response to infection of animals with HTLV-ill. The derivative, which is a polypeptide reactive with antiserum against t-3, is attached. and the reactivity of the sample toward tat-3 or tat-3 derivatives. Regarding a method for detecting infection of animals by HTLV-I[[ do.

All these embodiments of the invention, as well as other descriptions herein, can be readily accomplished and It is considered another aspect of the invention.

Detailed description of the invention HTLV-II [tat-3 protein] was extracted from E. coli in an easily recoverable amount. and that the protein thus expressed can be expressed in HTLV-III virus. It has frequently been shown that the virus is reactive with serum from animals infected by the virus. Served.

The E. coli expression vector of the present invention can be obtained by recombinant DNA methods or by recombinant D Prepared by a combination of NA and synthetic methods. It is at least HTLV- m tat-3 protein or tat that is immunologically equivalent to tat-3 -3 characterized by consisting of a coding sequence for a protein derivative Ru. The term "immunologically equivalent" means that the derivative polypeptide is Antibodies against standard tat-3 induced in response to infection of animals by In other words, it is reactive to standard tat-3. In the expression vectors of the invention, the coding sequence is operably linked to regulatory elements. be done.

The coding sequence for the specimen tat-3 was isolated from the virus mRNA. and cDNA preparation by reverse transcription from HTLV-nI virus or H It can be prepared from TLV-■ infected cells by a known method. It takes tat Preparation of the -3 coding sequence was described by Arya et al. al., 5science), 229.69 (1985') and Sodoro Ski et al., Science (5odroski et al., 5science) , 229.74 (1985), both of which are incorporated by reference. , which is incorporated herein by reference.

Prepared by semi-recombinant DNA and/or synthetic method Takeomote Showa 63-502000 (4) Wear. These are Botstein, Sci' ence), 229.1193 (1985). Contains. Such derivatives can be used to generate fused, mutated or truncated polynucleotides upon expression. 1 or more when the repeptide is immunologically equivalent to the standard tat-3 may consist of base pair additions, substitutions or deletions. Typically used as a diagnostic For example, such derivatives include truncated protein 1, such as N- or C-terminal amino acids. Immunological studies on tat-3, such as tat-3 proteins lacking amino acids, Polypeptides of 5 to 10 amino acids that maintain cross-reactivity with Most of the coding sequences are known, and none are available. can be inserted into an E. coli expression vector. The term “regulatory element” refers to an expression regulatory element. sequences, e.g. promoters and liposomes required for transcription and subsequent translation. refers to the binding site of the genome. Adjustable regulatory elements, i.e. non-configurable but inducing. Regulatory signals that require activation or derepression are preferred. Such a vector Ostensibly, in addition to regulatory elements, the vector is stably maintained in the host cell population. region, i.e., the replicon or translation start site and/or its The above selectable markers, i.e., a selectable phenotype in a host carrying the vector. Consists of genes that confer One example of an expression vector of the invention is Kula, Methods in Enzymology (Rosenberg et al., Meth, Enzym, ), 101.123 (1983) and Shirtrama N et al., In Experimental Manipulation of Gene Ex. Pression, edited by M. Inouuni, Academic Press, New York (Sha tzman et al,, in Experimental Manipul ation of Gene Express, 5sion, edit, by M.

Inouye, Academic Press, New York), 19 This is the plasmid pAs1 described in 82. pAsl is pBR322 replication Starting point, ampicillin resistance marker and PL, N-anti-termination function recognition site (NutL and NutR), rho-dependent transcription termination signals (tRl) and and its G residue are of the following formula: %formula% [In the formula, * indicates the cleavage site for BamHI, and! is cleavage for NdeI Show parts] Immediately following the BamHI cleavage site of Bacteriophage lambda consists of regulatory elements containing a genome binding site (RBS). It possesses a series of fragments.

pAsl is Bam in the lambda-pB R322 bond of pKc30cll. Deleting the nucleotide between the HI site and cnATG and recombining the molecule pKC30cI by regenerating a BamHI site immediately downstream of the ATG. It can be obtained from I. Is pKc30cII a lambda carrying the C■ gene? Inserted the 1.3k bHaem fragment from et al. into the HpaI site of pKc30. It consists of: Shatzmanet et al. b Said literature and Rosenberg et al. b See the above literature. pKc30 was determined by Shimatake et al., Nature (Shimatake et al. et al., Nature), 292.128 (1981). ing. )(indm and tetR gene of pB'R322) The 2.4 kb Hi nd I [-B pBR322 derivative with amHI fragment. Structure similar to pASI The structure is from Courtney et al., Nat. ure), 313.145 (1985) and Cotewin et al. (Kotewicz etll, Gene), 35.249 (1985 )It is described in. P L, Nut L, Nut R and c l1rb Derivatives of pAS1 consisting of s regulatory elements can be prepared by standard methods. Wear. the coding sequence functionally, i.e. in the correct orientation and proper It is ligated to the regulatory elements of the E. coli expression vector by standard methods in the reading frame. The expression vector of the present invention is prepared.

As shown in the examples below, tat-3 expressed by E. coli is For 42 of 92 samples (46%) of serum from individuals exposed to V-III. It was reactive, but not to serum from normal individuals. However, Therefore, tat-3 and its derivatives are % tat-3 protein or anti- Standard assays capable of detecting the presence of tat-3 antibodies Can be used for detection. the existence of more animal groups now or in the future Although not excluded, the known host range for HTLV-III includes humans and Restricted to certain other higher primates. Preferably, tat-3 is env, sor, gag or 3' or f gene products in serum used in one or more other test tools when performing an immunoassay for the presence of I can stay. Based on the data collected to date, a positive response to tat-3 is , HTLV-I [infection io.

%Diagnose. tat-3 derived from E. coli can also be used to scan blood samples from blood banks. It can be used for cleaning.

Methods for using tat-3 in such diagnostic immunoassays are well known. child These are, for example, Casay et al., Journal of Biology. tal,,J.

Virol, ), 55.417 (1985) and Crow et al. Disclosed in L et al., Ce1l), 41.979 (1985). This includes methods for tat-3 is a ux-linked immunosorbent assay (EL ISA) or radioimmunoassay (RIA). EL Preliminary experiments have shown that the ISA test produces a small percentage of false positives. HTLV-III, the virulence stimulates the production of antiserum that is reactive against HTLV-III. It can be used to intensify. therefore.

The tat-3 protein is thought to be localized in the cell nucleus rather than structurally. However, the protein is an antigen of vaccines against infection by HTLV-II [ Can be used as a component. The ability to activate polyclonal antibodies is also , Kohler and Milstein, Nature Stein, Nature), 256.495 (1975). monoclonal cells by standard methods described or other methods of cell fusion or transformation. Enables the production of local antibodies. Such polyclonal or monoclonal Local antibodies can also be used to detect the tat73 gene in cell populations, such as serum or cell cultures. Can be useful in detecting the presence of gene products. Such antibodies may also be used for DNA binding. tat-3 is localized in the functional region within the protein, such as the region where it acts. It is useful for affinity purification and epitope mapping of HTLV- It can be used as a reversal agent in the treatment of IIInl infections. The tat-3 gene product The organism is also described by Arya et al. and Sodorowski et al.

and 5 Odroski et al.), disclosed by the above-mentioned documents. It can then be used for the regulation of LTR-regulated gene expression units. tat -3 is functional in trans by reason. , the expression vectors of the present invention can be assembled into complete gene expression units or other plasmids. can be used to regulate expression for existing gene expression units. Ru.

The deduced amino acid sequence of the tat-3 gene product (see Example 1 below) was originally There are at least three recognizable regions in the 57 amino acids of region (5 of 16 residues from positions 2 to 18), cysteine-rich region (22 7 of 16 residues from position to position 37) and lysine/arginine rich region (8 out of 9 residues from positions 49 to 57) is a very hydrophilic protein. It shows good quality. Availability of sufficient amounts of highly purified tat-3 protein ability to directly elucidate its site and mechanism of action, e.g. its DNA binding properties. enable. Additionally, various truncated and mutated forms of this protein expression allows precise localization of its functional regions. tat derived from E. coli The availability of allows the identification of inhibitors of tat-3 function that can be used to

The tat-3 gene of HTLV-Ill is functionally linked to tat-1 and tat -2 genes and all %3 are about 2 kilobases from the 3 exons tat-3 is transcribed into the RNA of tat-1 and its partial nucleotide sequence. Different from 2.

Example Next, examples will be given to explain the present invention and how to implement and use the present invention. It does not limit the law.

Example 1 pOTs34 carries the transcription terminator-1oop terminator18 Insert the 9 base pair fragment into the NruI site downstream of the BamH1 site of pASl. and synthetic linker (Xba I, Xho I, Sac I) Insert into the 5all site between the BamHI site and the added terminator. obtained from pAsl. pOTs34 inserts the linker in the opposite orientation other than poTs-s or pOTsV (Devara et al. al.

Ce1l), 36.43 (1984)).

A vector carrying the cDNA for tat-3, pCV-1 (Arya et al. (Arya et al., supra) of the tat-3 coding sequence. used as a source. Nucleotide and deduced amino acid sequence of cDNA in pCV-1 The columns are shown below: ATG GAG CCA GTA GAT CCT AGA CTA GAG CCCTGG AAGMET GLU PROVAI, ASP PROARG LED GLU PROTRP LYSCAT CCA GGA AGT C AG CCT AAA ACT GCT T'GT ACCAATHls PR OGLY SERGLN PROLYS THRALA CYS THRASN TGCTAT TGT AAA AAG TGT TGCTIT CAT TG CCAA GTrCYS TYRCYS LYS LYS CYS CYS P HE　HIS　CYS　GLN　VALTGT　TTCATA　ACA　AAA GCCTTA GGCATCTCCTAT GGCCYS PHE ILE T) IRLYS ALA LEU GLY ILE SERTYRGLYAGG AAG AAG CGG AGA CAG CGA CGA AGA CCT CCT CAAARG LYS LYS ARG ARG GLN ARG ARG ARG PROPROGLNGGCAGT CAG ACT CAT CAA GTr TcT CT’A TCA AAG’CAAGLY SER GLN THRHIS GLN VAL SERLEU SERLYS GLN CCCACCTCCCAAA TCCCGA GGG GACCCG ACA G GCCCGPROTHRSERGLN SERARG GLY ASP PRO THRGLY PROAAG GAA TAG LYS GLU is over.

The method used to express the complete tat-3 protein involved two steps. . First, the first 12 base pairs (bp) are deleted (tat-3 copies) at its 5' end. The coding region was cloned with Mbol restriction endonuclease from the cDNA clone. Obtained as a ragment. This fragment was added to the pOTs34 vector using BamHI. inserted into the site. The resulting construct, pOTs-tatIIID, contains codons 2-4. was deleted and the start codon provided by pOTs34 was placed in-frame. tat-3 coding sequence. The second step is to This included regeneration of three missing codons. 5' BamHI site instead of 3' to pO After regeneration with the Ts-tallD plasmid, this vector was deleted with BamHI. The start codon and blunt-end cloning site immediately adjacent to the 50th codon of tat-3 was prepared. Next, synthetic NA that reconstructs the missing codon! Insert J car did. The nucleotide sequence of the linker is such that the second, third and fourth codons are as follows: tat without changing the amino acid sequence such as GAA CCGGTG. 3 genes were slightly modified. This structure consists of Bam between the fourth and fifth codons. It occurred at the HI site. The final structure, pOTS-tatII, is the AT of pOTs34. Played in-frame with G, including unomitted tat, -3 coding sequence . Samples of pOTs-tatm were shipped to American Tariff under the terms of the Budapest Treaty. Ip Culture Collection, Rocksville, Maryland Accession No. It has been deposited under No. 53305.

pOT5-tatn[D%pOTS-tatm and the pair without inserts vector (pOTs34), E. coli AR120, inducible with nalidixic acid. was introduced into a capable cI lithoan. Mott et al., Proceedings of the National Academy of Sciences, ott et al, Proc.

Nat’1. Acad, Sci, U.S.A.), 82.88 (1985 ).

Parts of the bacterial lysate were subjected to polyacrylamide gel electrophoresis at different times after induction. Ta. pOTs34, pOTS-tatmD or pOT5-tatI [[Containing AR120 bacterial cells were grown to 0D650 = 0.4-0.5, and the 60 py/in the same manner as described by Mott et al. rnl! induced by the addition of nalidixic acid up to O8, 3 and 5 after induction Take an aliquot, spin down and add lysis buffer (60mM) to Lys-HC1! ( pH 7.0), 10% glycerol, 5% 2-mercaptoethanol, 2% SD S, 0.1% bromophenol blue polyacrylamide gel (30:0.8 Coomassie brilliant ・Pull (Coomassie Brilliant Blue) R-250 It was stained and visualized.

The protein diffused with the 14kd lysozyme marker was specifically added to pOTS-ta. induced in cells transfected with tmD, producing a slightly larger protein. was detected in cells transfected with po'rs-tatm. 14kd view The molecular size is approximately 9.7 kd based on the amino acid sequence of tat-3. larger than the expected molecular size of This is due to the high proline content of the protein. This example is based on E. coli Figure 2 shows high level expression of the tat-3 coding sequence in the cells.

Example 2 Similar to Example 1, production of highly expressed tat-3 protein in bacteria ( 2-5% of total cellular proteins) can be produced by preparing specific polyclonal antibodies against it. made possible. For this purpose, after analysis on a prepared polyacrylamide gel, The tat-3 protein derived from E. coli purified by pneumatic elution is was injected subscapularly into white rabbits. Antisera from immunized rabbits are produced by bacteria. It reacted well with the expressed 14kd protein. In addition, polyacrylamide A protein of the same size, 14 kd by gel electrophoresis, was obtained by immunoprecipitation. Detected at low concentrations in infected T-lymphocyte cell line (H9/HTLV-Ill-B) . This latter data shows that even after SDS gel electrophoresis, tat- This is the first evidence that tat-3 has a common epitope with native tat-3. Therefore, bacterial-derived tat-3 is treated with an antibody that is reactive against standard tat-3. This shows that it can be used to produce the body.

Example 3 To investigate whether the tat-3 protein may have diagnostic or prognostic value. For this purpose, serum from various individuals was partially purified by Western blotting. We investigated the reactivity of the protein with the target protein. In particular, partial purification, 5DS-port After analysis on a lyacrylamide gel and electrotransfer to a nitrocellulose membrane, the membrane was Cut into strips and each strip was determined to contain tat-31μ2. the strip Milk buffer (5% nonfat dry milk, 0.1% antiform A, 0.1% N a N 3. Incubate in 0.9% NaC1) at room temperature for 1 hour, then incubate with patient blood. A 17,100 dilution of the supernatant was used and incubated at 4°C. Then, remove the strip. Wash for 20 minutes in salt buffered saline (PBS) containing I-labeled protein A. The milk buffer was incubated for 1 hour at room temperature (20-25°C). Each After washing three times for 30 min in PBS, the strips were dried and subjected to autoradiography. did. The 14.3 kd band corresponded to tat-3. The results shown in Table 1 below are , HTLV-I [[] All healthy subjects who are not recognized to have been exposed to HTLV-I Although it lacked antibodies to target other viral structural proteins (envelope and core), A substantial proportion of people who are seropositive for TAT-3 This indicates that the antibody concentration was as high as possible.

107 serum samples were examined. Place them in a generally accepted center Center for Disease Control Divided into four categories based on the definition of: (1) healthy seronegative (ga (HN) i2) Healthy HTLV-II I carriers (including serum from individuals in high-risk populations and env or g Positive for HTLV-III antibodies against ag protein, but with no clinical symptoms (3) individuals with AIDS-related complex (ARC); and (4) one patient with acquired immunodeficiency syndrome (AIDS).

category! None of the serum samples from J-(1) were reactive with tat-3. It was. In each of the other categories, approximately the same proportion of samples (53%, 29% and 53%) were reactive towards the tat-3.

The response varies from strong to weak reactivity without correlation to the stage of disease progression. did.

Table 1 % tat-3 lower immunity when compared to env or gag proteins The reaction is likely to be due to lower concentrations of the protein expressed in vivo, Due to the presence of fewer immunogenic epitopes and the putative nuclear localization of tat-3 do. This example demonstrates the use of bacterial sources in diagnosing infection with HTLV-III in animals. This shows the usefulness of the tat-3 protein.

The foregoing description and examples are illustrative of the invention and its preferred embodiments. death However, this invention is not limited to the specific examples specifically disclosed herein. and includes all modifications falling within the scope of the following claims.

international search report 1MI*’MIle++al^”””’-pcr/Is%n, 174

Claims (1)

[Claims] (1) consisting of a DNA coding sequence operably linked to a regulatory element; tat-3 protein whose coding sequence is HTLV-III or a derivative thereof which encodes a derivative that elicits a response to infection of animals with HTLV-III. The polypeptide is reactive with antiserum against tat-3. Characterized Ecoli expression vector. (2) The DNA coding sequence is an amino acid sequence: [There is a sequence] (wherein n means 0 or 1) Vector of term (1). (3) The DNA coding sequence has the following sequence: (wherein n means 0 or 1 and X means G or A) Vector of terms. (4) The regulatory element recognizes the PL promoter of lambda, the NutL and NutR and said cII ribosome binding site (1), (2) or Vector in term (3). (5) pAS1 or pAS1 into which the coding sequence for tat-3 has been inserted; The vector of item (1), (2) or (3) above, which is a derivative thereof. (6) the above-mentioned () which is pOTS-tatIIID or pOTS-tatIII; 5) Term vector. (7) Serum samples from animals and tat-3 or its derivatives are HTLV-III antiserum against tat-3 elicits a response to infection of animals with contacting the derivative which is a reactive polypeptide and contacting the tat-3 derivative with the tat-3 derivative. An animal using HTLV-III, characterized in that the reactivity of the sample is assayed for How to detect infection. (8) the tat-3 or tat-3 derivative is operably linked to a regulatory element; NA coding sequence, the DNA coding sequence is HTLV-III tat-3 protein or a derivative thereof, and the derivative encodes the HTLV-I against an antiserum against tat-3 that elicits a response to infection of animals with II. from E. coli transformed with an expression vector that is a polypeptide that is reactive with The method of paragraph (7) above. (9) The tat-3 or tat-3 derivative has the following amino acid sequence: [There is an array] (In the formula, n means 0 or 1) The method of paragraph (8) above. (10) The DNA coding sequence is the following [sequence] (wherein n means 0 or 1 and X means G or A) Section method. (11) In the vector, the regulatory elements include the PL promoter of lambda, NutL and and the NutR recognition site and the cII ribosome binding site. , (9) or (10). (12) the vector is a pA into which the tat-3 coding sequence has been inserted; The method of item (8), (9) or (10) above, which is S1 or a derivative thereof. (13) The vector is pOTS-tatIIID or pOTS-tatIII The method according to item (12) above. (14) Also, the sample and one or more other HTLV-III gene products (7), (8), (9) or (10), which consists of contacting Law. (15) Contacting the sample with the tat-3 in Western plotting assay The method of item (7), (8), (9) or (10) above.