| AIDS caused by HIV contraction is a serious infectious disease threatening the health of human beings and stability of society. It is increasingly important to develop effective HIV vaccines to prevent more people from contracting HIV and lower the morbility rate of infectors.HIV-1 Tat (trans-activator of transcription) is a key viral regulatory protein and is necessary for viral replication, spreading and pathogenesis. After infecting target cells, the Tat generated in cytoplasm can reach the nucleus and bind to the transactivating responsive region(TAR) to prolong the HIV-1 RNA. In addition, Tat is secreted from infected cells into extracellular medium to play an important role in pathogenesis:â‘ inhibit proliferation and differentiation of immune cells and induce their apoptosis;â‘¡activate static CD4+ T cells, contributing to HIV infection and spreading in vivo;â‘¢promote Kaposi sarcoma tumor cells growing;â‘£cause nerve injury, showing neurotoxicity. Blocking the influence on virus replication and ecto-toxic effect is the mainstay to develop the prophylactic and therapeutic Tat vaccines.However, many studies showed that the structure of native Tat was instable and the titer of antibody induced by native Tat was low. Both recombinant Tat and chemically synthetical Tat can not induce the desired protective antibodies both at home and abroad. Therefore, we modified Tat according to its structure and function to enhance the stability of its structure, immunogenicity to induce high-titer and long-lasting neutralizing antibody.Four parts for the study:1. Segmentative expression of HIV-1HXB2 Tat protein in E.coliTo analyse antibodies distribution induced by novel Tat immunogens and native Tat, we divided the Tat into five regions (1-48aa, 22-72aa , 38-101aa, 49-101aa and 60-101aa).Full-length tat, tat(1-48aa), tat(22-72aa), tat(38-101aa), tat(49-101aa) and tat(60-101aa) were obtained in vitro by PCR and cloned into pMD18-T vector by T/A cloning. Then the correct DNA sequences were inserted into pPEPTIDE prokaryotic expression vector. The six plasmids pPEPTIDE-tat, pPEPTIDE-tat(1-48aa), pPEPTIDE-tat(22-72aa), pPEPTIDE- tat(38-101aa), pPEPTIDE-tat(49-101aa), pPEPTIDE-tat(60-101aa) were constructed successfully and transformed into E.coli BL21(DE3). They were induced by IPTG and the relative molecular weight(MW) of expressed products were 34400 of Tat, 28800 of Tat(1-48aa), 29400 of Tat(22-72aa), 30700 of Tat(38-101aa), 29500 of Tat(49-101aa), 28000 of Tat(60-101aa). The six expressed products were purified by Ni-NTA column.The reactivity of full-length Tat-PPEPTIDE and five Tat peptides with anti Tat--PET32a serum from rabbits were testified by ELISA. Results showed that Tat-PPEPTIDE and the five Tat peptides could react specifically with anti-Tat serum, of which Tat-PPEPTIDE showed the strongest reaction, the second was Tat(1-48aa) and Tat(22-72aa); the reactivity of C-terminal with anti Tat--PET32a sera was lower, of which Tat(49-101aa) showed stronger reaction thanTat(38-101aa) and Tat(60-101aa). The results indicate that the first exon of Tat containing basic region is important epitope . Tat-PPEPTIDE and the five peptides accurately reflected the antibodies distribution induced by the native Tat , which can be used in the analysis of other spectrums of antibodies induced by novel Tat immunogens.2. Expression and immunogenicity analysis of HIV-1 HXB2 strainTat(B41-101N) and Tat(B41-101C) fusion proteins Many studies showed that basic region (49-57aa) of Tat mediated cell-penetrating which closely related to Tat extracellular activity and C-terminal region(73-101aa) of Tat contained RGD motif which closely related to its toxicity. Based on the above findings, we added Tat41-101aa before N-terminal of native Tat to construct Tat(B41-101N) and replaced the N-terminal with Tat41-101aa to construct Tat(B41-101C) to induce higher titer of neutralizing antibodies which can bind basic region and C-terminal region of Tat.The primers were designed according to the Tat sequence of HIV-1 HXB2 strain and the preferred codons of E.coli. The tat(B41-101N) and tat(B41-101C) were synthesized in vitro by overlapping PCR and sequenced after inserted into pMD18-T vector by T/A cloning. Then the correct tat(B41-101N) and tat(B41-101C) were respectively inserted into pET32a vector to construct prokaryotic expression plasmids pET32a-tat(B41-1011N) and pET32a-tat(B41-1011C) . The two recombinant plasmids were transformed into E.coli BL21(DE3) for expression The fusion protein Tat(B41-101N) and Tat(B41-101C) were expressed with respective relative molecular weight(MW) 36000, 34000 under induction of IPTG, purified by Ni-NTA column and testified with specific anti-Tat monoclonal antibody by Western-blot. Rabbits were vaccinated with Tat(B41-101N), Tat(B41-101C) and native Tat. The titers of sera were testified by ELISA. The result showed that the titers of antibodies induced by Tat(B41-101N), Tat(B41-101C) and native Tat were respectively 200000, 1600000, 800000. Testify the reactivity of sera with the abovementioned five Tat peptides by ELISA. Results showed that: (1) the reactivity of anti-Tat(B41-101N) and anti-Tat(B41-101C) antisera with Tat(1-48aa) were significantly lower than the native Tat, indicating that Tat(1-48aa) depends on the conformation of native Tat; (2) the reactivity of three types of sera with Tat(22-72aa) were all strong, indicating that Tat(22-72aa) are more stable Tat epitope; (3) Tat(B41-101C) induced higher anti-Tat(49-101aa) and anti-Tat(60-101aa) antibody than native Tat, showing the different immunogenicity from native Tat, which is important for the improvement of Tat vaccine; (4) Tat(B41-101C) induced higher anti-Tat(49-101aa) and anti-Tat(60-101aa) antibody than Tat(B41-101N), indicating that N-terminal region probably inhibits C- terminal region from inducing antibody.To conclude, we successfully constructed and expressed Tat(B41-101N) and Tat(B41-101C) fusion proteins, which showed different immunogenicity from native Tat. It laid the foundation on further study of novel Tat vaccines.3. Analysis of humoral immune responses induced by other novel Tat immunogensTo study the characteristics of different novel Tat immunogens more deeply to screen the best candidate vaccine, we designed other Tat immunogens:â‘ We deleted Cys-rich region(22-37aa) to construct Tatqs to enhance the stability ;â‘¡Many studies proved that Cys-rich region showed the function of auto-adjuvant for native Tat immunogen.To analyse the impact of position of Cys-rich region on immunogenicity, we put the region before N-terminal of native Tat to construct TatCN and after C-terminal of native Tat to construct TatCC;â‘¢The second part of our study showed that N-terminal region probably inhibited C-terminal region from inducing antibody. So we delete N-terminal region to construct Tat(22-72aa) which mainly contains the first exon of Tat and Tat(38-101aa) which mainly contained C-terminal region of Tat.Rabbits were immunized by native Tat, Tatqs (delete cys-rich), TatCC (put cys-rich after C-terminal region), TatCN (put cys-rich before N-terminal region), Tat(38-101aa) (delete N-terminal and cys-rich), Tat(B41-101N), Tat(B41-101C). Sera were collected and detected by ELISA. Rasults showed: (1) All Tat immunogens could induced specific anti-Tat antibodies , however, The titers of antibodies showed apparent differences : Tat(B41-101C) induced the highest level of antibody, the second was Tat and Tat(22-72aa) , TatCN was the lowest; (2) the reactivities of sera induced by all novel Tat immunogens with Tat(1-48aa) were significantly lower than the native Tat, indicating that Tat(1-48aa) depends on natural Tat conformation ; (3) the reactions of all types of sera to Tat(22-72aa) were strong, indicating that Tat(22-72aa) are more stable Tat epitope; (4) Tat(B41-101C),Tat(38-101aa) and TatCC induced higher anti-C-terminal antibodies than nativr Tat: Tat(B41-101C),Tat(38-101aa) induced higher anti-Tat(49-101aa) and anti-Tat(60-101aa) antibody, TatCC induced the highest anti-Tat(38-101aa) antibody among all immunogens. showing the different immunogenicity from native Tat, which is important for the improvement of Tat vaccine; (5) Tat(B41-101C) and Tat(38-101aa) induced higher anti-Tat(49-101aa) and anti-Tat(60-101aa) antibody than Tat(B41-101N) and Tatqs, indicating that N-terminal region probably inhibited C- terminal region from inducing antibody; (6) Deleting Cys-rich region( Tatqs) enhanced the stability, however, Tatqs, TatCC and TatCN induced lower level of antibody than native Tat, indicating the function of anto-adjuvant of Cys-rich region depends on the conformation of native Tat.To conclude, different novel Tat immunogens showed different immunogenicity from native Tat ,which laid the foundation on development of effective novel Tat vaccines and played an important role in understanding of the structures and functions of Tat.4. The comparision for the reactivity of sera from HIV-1 infectors with different Tat antigensDetecting the reactivity of sera from HIV-1 infectors with prokaryotic expressed native Tat, seven modified Tat protein ( Tat(B41-101N), Tat(B41-101C), Tat(B41-61N), Tat(B41-61C), TatCC, TatCN, Tatqs) and five Tat peptides (Tat(1-48aa), Tat(22-72aa), Tat(38-101aa), Tat(49-101aa), Tat(60-101aa) ) to compare their positive ratio and specificity of the reactivity. Our aim is to screen the best antigen to be used in the clinical detection.ELISA showed : (1)there were three modes of the reaction of sera from HIV patients to different Tat antigens :â‘ Both the proportion and OD values of the sera which showed positive reaction to a variety of antigens containing Tat(41-61C) were the highest ;â‘¡The proportion of the sera which mainly showed positive reaction to Tat (22-72aa) was lower than the proportion of the first mode, OD values were the lowest;â‘¢the proportion of the sera which mainly showed positive reaction to TatCN was lower (only two) ,but OD values were higher. Above study suggested that different Tat proteins or peptides showed different reactions to sera from HIV patients. Moreover, , clinical research had shown that anti-Tat antibodies from HIV patients could slowen HIV process .The study on the three response modes laid the foundation on classifying HIV patients ,analyzing the relevance between the types of sera and process of disease and played an important role in the improvement of novel Tat vaccines; (2) The OD values and positive rates detected by different antigens showed apparent differences , many novel antigens are better than native Tat , Whether these antigens are complementary, it is worth further study ; (3) The reactions of Tat(B41-101C) and Tat(B41-61C) to HIV sera were stronger than Tat(B41-101N) and Tat(B41-61N) , revealing N-terminal region probably blocked C-terminal region binding to antibody in serum .To conclude, the different Tat antigens for detecting showed some similarities and characteristics which laid the foundation for the further study of the Tat antigens and antigens screening for clinical use.Summary:1. Full-lengh Tat and five peptides were successfully expressed and accurately reflected the antibodies distribution induced by the native Tat , which can be used in the analysis of other spectrums of antibodies induced by novel Tat immunogens.2. Seven novel Tat immunogens were ere successfully constructed and expressed. Different novel Tat immunogens showed different immunogenicity from native Tat ,which laid the foundation on development of effective novel Tat vaccines and played an important role in understanding of the structures and functions of Tat.3. We used five Tat peptides and different modified Tat proteins to detect anti-Tat antibody in sera from HIV patients. Results showed that different Tat proteins or peptides had different reactivities, which played an important role in the analysis of the relevance between the types of sera and process of disease, and laid a foundation on development of effective novel Tat vaccines and the high-specific and sensitive antigens for detecting anti-Tat antibody.
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