Construction, Immunogenicity And Antigenicity Analysis Of Recombinant Tat Mutant Proteins Of Human Immunodeficiency Virus Type I

Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome caused by HIV, which greatly undermine people's health, are big health problems faced by both China and other countries all over the world. It is of great importance to control HIV infection and AIDS epidemic by developing safe and effective HIV vaccine or anti-HIV medicine.Trans-activator of transcription (Tat) is an important regulatory protein of HIV-1 which plays an important role in HIV-1 replication and infection, and is produced at an early stage after HIV-1 infection. In HIV-1 infected cells, Tat can trans-activate HIV-1 genome to initial its transcription and elongation, thus promote HIV-1 replication. Besides, extracellular Tat protein secreted by infected cells exerts some other multiple functions as in immunosuppression, AIDS-related brain damage and Kaposi carcinoma and is called a kind of viral toxin, which makes it a likely candidate antigen in HIV vaccine development.It is reported that even though Tat is conserved in amino acids sequences among different isolates, it doesn't have a fixed construction, which makes it difficult for native Tat to induce high level antibody with immunoprotection. According to the construction of native Tat protein and the functions of different regions of it, we have constructed many prokaryotic expression plasmids which were induced to express in E.coli to produce recombinant mutants in order to improve its stability, immunogenicity and finally to induce high level antibody production.The whole study consists of three parts as follows:PartⅠ: Prokaryotic Expression of Tat Truncated Mutants of HIV-1 HXB2 IsolateBased on the functions of each region of Tat protein (N terminus (1-21aa), cysteine-rich region (22-37aa), the core region (38-48aa), the basic region (49-59aa), glutamine-rich region (60-72aa) and C terminus (73-101aa)) and our previous research, nine truncated Tat proteins from HIV-1 HXB2 isolate were constructed in this study. The methods used are as follows: DNA fragments of tat1-21, tat1-37, tat1-61, tat1-72, tat1-86, tat22-86, tat38-61, tat22-101(a), tat22-101(b) were obtained through PCR and were cloned onto vector pMD18-T. After being sequenced, the correct ones were cloned onto expression vector pET-32a or pPEPTIDE2 to construct prokaryotic expression plasmids: pPEPTIDE2-tat1-21, pPET-32a-tat1-37, pPET-32a-tat1-61, pPEPTIDE2-tat1-72, pPEPTIDE2-tat1-86, pPEPTIDE2-tat22-86, pPEPTIDE2-tat38-61, pPET-32a-tat22-101 and pPEPTIDE2-tat22-101. Then all the plasmids were transformed into E.coli BL21(DE3) and induced to express by IPTG. Nine recombinant proteins PEPTIDE2-Tat1-21, PET-32a-Tat1-37, PET-32a-Tat1-61, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86, PEPTIDE2-Tat22-86, PEPTIDE2-Tat38-61, PET-32a-Tat22-101 and PEPTIDE2-Tat22-101 were purified by affinity chromatography with the molecular weight 25400, 22000, 23100, 31300, 33200, 26900, 31100, 26400 and 32600 respectively.Western blot assay showed that five mutants containing N terminus (PEPTIDE2-Tat1-21, PET-32a-Tat1-37, PET-32a-Tat1-61, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86) reactivated specifically with anti-Tat N terminus monoclonal antibody, mutants lacking of N terminus and two vector proteins PET-32a and PEPTIDE2 didn't bind to anti-N terminus monoclonal antibody. And ELISA assay showed that four mutants PEPTIDE2-Tat22-86, PEPTIDE2-Tat38-61, PET-32a-Tat22-101 and PEPTIDE2-Tat22-101 reactivated well with anti-full-length Tat rabbit serum and no reactivity was detected with PET-32a and PEPTIDE2. These demonstrated that nine recombinant Tat mutants were successfully constructed in this study.PartⅡ: Immunogenicity Analysis of Recombinant HIV-1 Tat MutantsFour truncated recombinant Tat mutants PET-32a-Tat1-37, PET-32a-Tat1-61, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86 (with N terminus in each mutant) and recombinant full-length Tat1-101 were used to immunize New Zealand rabbits to raise antisera to determine their ability of inducing the production of anti-N terminus antibody. A mutant PET-32a-Tat22-101 without Tat N terminus and PET-32a-Tat1-101 were used to immunize New Zealand rabbits and BALB/c mice to compare their abilities of inducing antibodies to C terminal region of Tat.The results of ELISA showed that rabbit and mouse antisera raised by recombinant Tat mutants described above and full-length Tat reactivated specifically with full-length Tat protein, which demonstrated that the mutants constructed in this study preserved its immunogenicity. However, recombinant Tat mutants containing Tat N terminal region (PET-32a-Tat1-37, PET-32a-Tat1-48, PET-32a-Tat1-61, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86) induced low level antibody (titers between 200-3200) against chemical-synthesized peptide Tat1-21 (sTat1-21), compared with recombinant full-length Tat PET-32a-Tat1-101 which induced relatively high level antibody against sTat1-21 in rabbits with the titer reaching 25600, suggesting that it might be at a conformational dependent manner in inducing anti-N terminus antibody and only native full-length Tat can maintain the best conformation to induce anti-N terminus antibody. Secondly, we investigated the pattern of antibody production induced by PET-32a-Tat22-101 and found that the rabbit and mouse antisera raised by PET-32a-Tat22-101 showed stronger reactivity with Tat C terminal antigens PEPTIDE2-Tat38-101, PEPTIDE2-Tat49-101 and PEPTIDE2-Tat60-101, which suggested that Tat N terminal region might suppress the production of antibodies against other epitopes.PartⅢ: Preliminary Antigenicity Analysis of Recombinant Tat MutantsFive recombinant Tat mutants PEPTIDE2-Tat1-21, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86, PEPTIDE2-Tat22-86 and PEPTIDE2-Tat22-101 were used to detect antibodies by ELISA in rabbit antisera which were raised by eight different Tat mutants and collected previously. The eight kinds of rabbit antisera were anti-PET-32a-Tat1-101, PET-32a-Tat22-72, PET-32a-Tat38-101, PET-32a-TatCC, PET-32a-TatCN, PET-32a-TatΔC, PET-32a-TatB41-101C and PET-32a-TatB41-101N antisera.The results showed that PEPTIDE2-Tat1-21 showed the strongest reactivity with anti-PET-32a-Tat1-101 antiserum, compared with other rabbit antisera, which demonstrates that N terminal region is an important epitope recognized by anti-Tat1-101 antibodies. Besides, two Tat N terminus deleted mutants PEPTIDE2-Tat22-86 and PEPTIDE2-Tat22-101 showed different reactivity patterns with rabbit antisera tested and reactivated well with most of the antisera, suggesting that epitopes of Tat mutants changed compared with native Tat antigen.Further, we detected human sera samples (anti-Tat positive) from ten AIDS subjects with native recombinant full-length Tat PET-32a-Tat1-101 and its 7 recombinant mutants PET-32a-Tat1-48, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86, PEPTIDE2-Tat38-61, PEPTIDE2-Tat38-101, PEPTIDE2-Tat49-101 and PEPTIDE2-Tat60-101. The results of ELISA assay showed that (1) the same serum bound to different Tat mutants with different avidity, and PEPTIDE2-Tat1-86 showed the strongest avidity with all of the sera, (2) the same Tat mutant showed different sensitivities to different sera, which deserve further investigation due to general characteristics as well as specialties.Summary:1. In this study, we successfully expressed nine truncated proteins of Tat from HXB2 isolate by constructing prokaryotic expression plasmids which were transformed into E.coli.2. Five Tat mutants (PET-32a-Tat1-37,PET-32a-Tat1-61,PEPTIDE2-Tat1-72,PEPTIDE2-Tat1-86 and PET-32a-Tat22-101 ) preserved its immunogenicity and induced antibodies of different characteristics as native recombinant Tat protein did, especially PET-32a-Tat22-101 which raised antiserum showing stronger reactivity with recombinant protein lacking N terminal region than anti-PET-32a-Tat1-101 antiserum, suggesting that the N terminus within native Tat protein suppresses the induction of antibody against other epitopes. This result is of great significance for the structure and function study of Tat protein, moreover, it lays a foundation for the development of novel Tat vaccine.3. In this study, we found that among all the recombinant proteins, only recombinant full-length Tat protein induced relatively high level of antibody against N terminus and truncated proteins induced much lower level of antibody against the region, suggesting that anti-N terminus antibody was induced at a conformational dependent manner.4. Preliminary study showed that native full-length Tat and its truncated mutants reactivated with sera of AIDS patients with different sensitivities and magnitudes, especially Tat1-86 which reactivated well generally with all the sera detected in the study, providing clues for further study in analyzing and screening efficient antigens for clinical detection.