Reconstruction Of Tat Protein Of Human Immunodeficiency Virus Type1and Preparation Of Tat-based Particular Antigens

Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) 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-1which plays an important role in HIV-1replication and infection, and is produced at an early stage after HIV-1infection. In HIV-1infected cells, Tat can trans-activate HIV-1genome to initial its transcription and elongation, thus promote HIV-1replication. 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 has conservation of 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 hydrophobic property of native Tat protein and the functions of different regions of it, we have constructed different HIV-1Tat mutants and its particulate recombinant antigen fused with high hydrophobic region (122-191aa) of hepatitis C virus core protein (HCVC), prepared Tat and its mutant proteins labeled with colloidal gold respectively and immunized C57BL mice, expressed Tat-HCVC122-191recombinant antigens in cell-free protein synthesis system and identified their particulate characteristics by detection using negative staining transmission electron microscope. The results in this study might provide some experimental basis for further study of HIV Tat functions as well as for development of potential safe and effective Tat particulate immunogens.The whole study consists of three parts as follows:Part I:Prokaryotic expression of the recombinant antigens of HIV-1Tat mutants fused with HCV core proteinHIV-1Tat protein contains six functional regions including N terminus (1-21aa), cysteine-rich region (22-37aa), the core region (38-48aa), the basic region (49-57aa), glutamine-rich region (60-72aa) and C terminus (73-101aa)). Base on our previous studies on reconstruction of Tat and analysis of Tat immunogenicity, in this study, we analyzed the hydrophobic characteristics of Tat protein, constructed and prokaryotic expression HIV-1HXB2Tat mutant fusion protein PET32a-TatA(31-45) which deleted Tat hydrophobic region (31-45aa). The methods used are as follows:DNA fragment of tatl-101Δ(31-45) was obtained by PCR and overlap PCR by using plasmid template of pPEP-Tat1-101-HCVC(122-191), and cloned onto prokaryotic expression vector pET32a. After being sequenced, the correct ones was named pET32a-TatA(31-45). Then the plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The expression of PET32a-TatA(31-45) protein obtained in this study accounted for about55%of the total bacterial protein, which was significantly higher than that of the full-length Tat and other Tat mutant proteins. PET32a-TatA(31-45) protein was purified by affinity chromatography with the molecular weight27,600. In addition, previously HIV-1Tat recombinant plasmids (pET32a-Tat1-101, pET32a-Tat22-86, pET32a-Tat-22-101, pPEPTIDE2-Tatl-21, pPEPTIDE2-Tat38-61and pPEPTIDE2-Tat60-101) constructed correctly in our laboratory were expressed respectively as above for using as testing and control proteins. Western blot assay showed that both PET32a-Tat1-101and PET32a-TatΔ (31-45) reactivated specifically with anti-Tat monoclonal antibody, vector proteins PET-32a didn’t show the reactivity.On this basis, high hydrophobic HCVC122-191with self-assembled characteristic was used as vector in this study to prepare the recombinant antigens of different Tat mutants fused with HCVC122-191respectively. After E. coli favorite codons of HCVC122-191gene were optimized by PCR, tatΔ (31-45) and HCVC(122-191) DNA fragment was spliced by overlap PCR and cloned onto vector pPEPTIDE2to construct prokaryotic expression recombinant plasmid pPEPTIDE2-TatΔ(31-45)-HCVC(122-191) correctly. The recombinant plasmid and previously constructed two kinds recombinant plasmids (pPEP-Tat1-101-HCVC(122-191) and pPEP-Tat22-86-HCVC(122-191)) were transformed into E. coli BL21(DE3) and induced to express by IPTG. However, in both inducement conditions (37℃for3.5h and30℃for10h) and by12%SDS-PAGE analyses, the expression of the plasmids did not reach expected results.Part II:Preparation of Tat and its mutant particular antigens labeled with colloidal gold and their immunogenicity analysisIn order to solve the problem that expression of the fusion protein of Tat with HCVC122-191did not reach expected results by prokaryotic expression system, in this study Tat and its mutant particles labeled with colloidal gold were prepared (Part II) and the Tat-HCVC122-191fusion particulate antigens were expressed by cell-free protein synthesis system (Part III). In this part, colloidal gold solutions were prepared firstly by using reduction method with trisodium citrate. Two gold particles with good dispersion and uniformity were obtained (average particle size of75.5nm and32.5nm, respectively). Tat and three Tat mutant fusion proteins (PET32a-TatΔ(31-45), PET32a-Tat22-101and PET32a-Tat22-86) were used to label75.5nm colloidal gold particles, and then the stabilities of the Tat-gold particles were detected by NaCl coagulation assay at different pH conditions. Above Tat and Tat mutant proteins labeled with colloidal gold particles were used to immunize C57/BL mice respectively. Titers of the mouse antisera and their reaction with different Tat peptides were detected by ELISA.The results showed that the Tat1-101, TatΔ(31-45), Tat22-101and Tat22-86protein labeled colloidal gold were able to maintain stability within the range of pH5.5to9.5, pH5.5to11.0, pH4.0to9.5and pH4.5to9.5, respectively. The results of ELISA showed that all experimental group could all induce mouse antisera against full-length Tat, of which, titer of anti-Tat sera induced by Tat1-101colloidal gold group was the highest (1,024,000) that was significantly higher than the Tat1-101protein group and the other groups; anti-Tat titer induced by TatΔ(31-45) protein raised significantly when the protein was labeled with colloidal gold, indicating that the immunogenicities of full-length Tat and TatΔ(31-45) protein could be enhance obviously by labeling colloidal gold.Preliminary epitope analysis showed that full-length Tat1-101protein labeled with colloidal gold was mainly induced anti-Tat N terminal epitope antibody; reactivity of both mouse antisera induced by Tat22-86protein and Tat22-86labeled with colloidal gold with peptides Tat38-61and Tat60-101respectively reached that of anti-full-length Tat antisera with Tat38-61and Tat60-101; reactivity of mouse antisera induced by Tat22-101labeled with colloidal gold with Tat C terminal Tat60-101improved significantly compared with that of anti-full-length Tat antisera; TatΔ (31-45) labeled with colloidal gold could induce similar higher level antibodies against peptide Tatl-21, Tat38-61and Tat60-101compared with Tat protein or Tat labeled with colloidal gold. The results demonstrated that the three kinds of colloidal gold-labeled Tat mutant fusion protein particles (PET32a-TatΔ(31-45), PET32a-Tat22-101and PET32a-Tat22-86) prepared in this study all retained good immunogenicities and could induce antibodies against different functional region epitopes of Tat, which lay the foundation for combined application of different Tat epitopes antigens to develop novel HIV-1Tat vaccine.Part III:Cell-free expression in vitro and identification of particulate fusion antigens of HIV-1Tat with HCVC122-191In this part, HIV-1Tat particulate antigens fused with HCVC122-191were expressed by cell-free protein synthesis system. The methods used are as follows: HCVC122-191DNA fragment was spliced with tat1-101, tat1-86, tat22-101, tat22-86and TatΔ(31-45) respectively by overlap PCR, which were cloned onto cell-free expression vector pIVEX2.4d after being identified by two restriction enzyme digestion and sequencing. Five cell-free expression plasmids (pIVEX2.4d-Tat1-101-HCVC122-191, pIVEX2.4d-Tatl-86HCVC122-191, pIVEX2.4d-Tat22-101-HCVC122-191, pIVEX2.4d-Tat22-86-HC VC122-191and pIVEX2.4d-Tat A(31-45)-HCVC122-191) were constructed and expressed in cell-free protein synthesis system based on E. coli cell lysis buffer. The expressed HIV-1Tat-HCVC122-191recombinant proteins were identified by12%SDS-PAGE and Western blot, and their particulate characteristics were detected by using transmission electron microscope.The results showed that two expressed products of recombinant proteins (Tat22-101-HCVC122-191and Tat22-86-HCVC122-191) were obtained with the molecular weight16.10kDa and14.35kDa respectively as expected. Western blot assay showed that the fusion proteins (Tat22-86-HCVC122-191) and Tat22-100-HCVC122-191) reactivated specifically with anti-Tat monoclonal antibody (anti-HIV-1Tat antibody (71-81). Furthermore, the two Tat-HCVC recombinant proteins (Tat22-86-HCVC122-191) and Tat22-100-HCVC122-191) displayed particulate characteristics obviously identified by using negative staining transmission electron microscope, which average diameter was175±30nm and188±39nm respectively, while the control group proteins (Tat22-86and Tat22-101) didn’t form particle-like structure, indicating that HCVC122-191with high hydrophobicity maintained good self-assemble function and could make novel Tat particulate antigens formed when Tat mutant proteins were fused with it. Summary:1. In this study, we successfully expressed a Tat mutant recombinant protein PET32a-TatΔ(31-45) by constructing a truncated Tat prokaryotic expression plasmid which hydrophobic region31-45aa of Tat was deleted, suggesting that the hydrophobic region of Tat may inhibit the expression of HIV-1Tat and its mutants.2. Three kinds of prokaryotic recombinant expression plasmids containing DNA sequences of Tat and high hydrophobic region (122-191aa) of HCV core protein (HCVC)(pPEPTIDE2-TatΔ(31-45)-HCVC122-191, pPEP-Tat1-101-HCVC122-191and pPEP-Tat22-86-HCVC122-191) were constructed correctly identified by restriction enzyme digestion and sequencing. The plasmids were transformed into E. coli BL21(DE3) cells, however, the expression of those did not reach expected results by conventional IPTG inducement (37℃for3.5h) and by low temperature IPTG inducement (30℃for1Oh).3. The immunogenicities of full-length Tat and TatΔ(31-45) could be improved obviously by colloidal gold labeling; the three kinds of colloidal gold-labeled Tat mutant fusion protein particles (PET32a-TatΔ(31-45), PET32a-Tat22-101and PET32a-Tat22-86) all retained good immunogenicities, of which TatΔ(31-45) labeled with colloidal gold could induce similar higher level antibodies against three Tat peptides (Tat1-21, Tat38-61and Tat60-101) compared with Tat protein or Tat labeled with colloidal gold, reactivity of both mouse antisera induced by Tat22-86protein and Tat22-86labeled with colloidal gold with peptides Tat38-61and Tat60-101respectively reached that of anti-full-length Tat antisera with Tat38-61and Tat60-101, and reactivity of mouse antisera induced by Tat22-101labeled with colloidal gold with Tat C terminal Tat60-101improved significantly compared with that of anti-full-length Tat antisera, indicating that colloidal gold-labeled Tat mutant protein particles could induce stronger antibodies against different functional region epitopes of Tat which might have the role of blocking the biological activities including transcriptional trans-activation of native Tat protein.4. Five cell-free expression plasmids (pIVEX2.4d-Tatl-101-HCVC122-191, pIVEX2.4d-Tat1-86HCVC122-191, pIVEX2.4d-Tat22-101-HCVC122-191, pIVEX2.4d-Tat22-86-HCVC122-191and pIVEX2.4d-Tat A(31-45)-HCVC122-191) were constructed and successfully obtained two novel Tat particulate antigens (Tat22-101-HCVC122-191and Tat22-86-HCVC122-191) in cell-free protein synthesis system. The results might provide some experimental basis for further study of HIV-1Tat functions as well as for development of potential safe and effective Tat-based HIV-1Tat vaccine, and also reveal that HCVC122-191could be used as a promising displayed vector which might provide novel approach for studies on particulate vaccines of other pathogens.