Construction Of HIV-1 Tat Core And Basic Region Mutation Libraries And Affinity Screening

Human immunodeficiency virus (HIV) is attributed to the slow Retroviridae virus, spreading mainly through blood, sexual contact or mother to child such as vertical channels, which can cause Acquired Immune Deficiency Syndrome (AIDS). At present, HIV infection has become a major public health problem which has to be faced by the world. Developing an effective HIV vaccine is an important means of prevention and control of AIDS. Therefore, it is very important for exploring new strategies and finding new vaccine targets on the success of the vaccine research and development.HIV-1 Tat (trans-activator of transcription) is a key viral regulatory protein emerged in the early stage of HIV infection, and is necessary for viral replication, spreading and pathogenesis. After infecting target cells, the Tat generated in cytoplasm can reach the nucleus and bind with some Transcription factors to form the transcription initiation complex (TIC), which can promote transcription, extension and replication of the viral RNA. In addition, Tat can also be secreted from infected cells into the extracellular with a variety of ways in order to play a "virus toxins" role:①playing an immunosuppressive function By inducing CD4+T cells, NK cells or apoptosis B cells;②promoting the formation of Kaposi's sarcoma (KS) through some ways,such as activating HHV-8 replication via JAK/STAT signal transduction, or binding the RGD (Arg-Gly-Asp) withαvβ3 andα5β1 integrins of the endothelial cells;③promoting the incidence of HIV encephalopathy through affecting calcium flow in the nerves cells or inducing the expression of TGF-β, TNF and other neurological toxic substances.Tat basic amino acid-rich region (49-57aa) is the base sequence of functioning the trans-membrane and nuclear localization, and is also one of the main neutralization epitope. Its neutralizing antibody can eliminate the penetration of other cells, which can prevent the occurrence of AIDS and immune suppression. In addition, Tat basic region sequences highly conserved among the various subtypes, which is conducive to creating a broader cross-reactivity antibodies. There is a study having demonstrated that the basic region epitope was not destroyed, and the activity of Tat trans-membrane and extracellular activity was greatly reduced even Tat51/55 site-directed mutagenesis. In This study we construct and screen of Tat basic region using phage display technology platform, and we have maintained the Tat core region (38-48aa) in order to ensure the integrity of epitopes of the Tat basic region, and build the Tat core and basic region mutant peptide fragment library connected by random Linkers, and then screen the library using anti-Tat rabbit serum in order to obtain the original sequence of novel immunogen. We want to get a range of the Candidate mutant sequence which have conformational epitope stability and significantly decreased biological activity. It's a basic research for Tat novel vaccine research and development.1. Expression and immunogenicity analysis of HIV-1 PET32a-Tat38-61proteinFunctional studies have shown that Tat protein core area (38-48aa) induce apoptosis by combinating uninfected cells tubulin and basic region (49-57aa) is playing an important role for trans-membrane function and extracellular activities mediated by Tat. Structural studies have shown that:Tat is a natural non-folded protein, and its concept is extremely unstable, so it's not an ideal antigen. It's very important for the phage library affinity screening whether Tat core basic region (CBR) maintain an important epitope mutations or not. Therefore, The Tat basic region of 38-61 fragment was built into the prokaryotic expression vector, then we purified PET32a-Tat38-61 protein and tested their immunogenicity in order to analyze whether this important epitope is retained.The primers were designed according to the HIV-1 Tat sequence and the preferred codons of E.coli. The tat38-61 were synthesized in vitro by PCR and sequenced after inserted into pMD18-T vector by T/A cloning. Then the right tat38-61 was inserted into pET32a vector to construct prokaryotic expression plasmids pET32a-tat38-61. The recombinant plasmid was transformed into E.coli BL21(DE3) for expression.The protein PET32a-Tat38-61 was expressed with relative molecular weight(MW) 21300 under induction of IPTG, purified by Ni-NTA column and testified with Two different anti-Tat rabbit serum and HIV-positive serum by ELISA. The results showed that:①there is a specific reaction and the reactivity between rabbit anti-PEPTIDE-Tat serum and PET32a-Tat38-61.The result shows that much lower than the native Tat, and A weak response with carrier protein PET32a.②HIV positive serum in 12 cases, there are four cases clear specific response with PET32a-Tat38-61, and 6 case specific response with positive control full protein PET32a-Tat1-101, and includes all the four cases and PET32a-Tat38-61 showed a specific response to HIV in serum. these results suggest PET32a-Tat38-61 kept Complete Tat protein basic region epitope.To conclude, we successfully constructed and expressed PET32a-Tat38-61 protein, and its'neutralization epitope of Tat basic region has been preserved, which laid the foundation on getting the antibodies against the basic region and screening phage display library.2. Construction of HIV-1 Tat Core and Basic Region mutant libraryStudies have shown that after two important sites(51K and 55R)of the Tat basic region are mutated, the neutralization epitop is not destroyed, and the activity of Tat trans-membrane and extracellular toxicity disappeared or were greatly reduced. Therefore, in order to obtain the mutants which have a stable epitope conformation and significantly decreased biological activity, we first constructed for the Tat core and basic region mutant library. In addition, construction of conformable large molecule mutation library is pivotal for molecular evolution study in vitro. In order to extend the range of the screening region of amino acid in Tat basic region gene, the 51K and 55R were mutated randomly by overlap PCR amplification with the random nucleotide primers. At the same time we add to six random linker X6 after the basic region 38-61aa, which not only expand the library of variants, but also help to generate a stable conformation of the novel immunogen.Through four rounds of PCR we got the tat38-61 (51X/55X) mutant fragments, and cloned into phage display vector pCANTAB5S-LD3 after digested by XbaⅠ. then we constructed HIV-1 Tat Core and Basic Region(TCBR) mutant library.The phage displayed library was generated by M13K07 rescue. After detection we found that as much as 5.0×106 clones were obtainded in the phage library and the titer was 2.65×1012 TU/ml. About 70% clones contained inserted Tat basic region 38-61(51X/55X) fragments. Sequence analysis of 24 samples showed that nucleotide acids and amino acids at randomized 51X-55X sites distributed randomly.In summary, we have successfully constructed HIV-1 Tat Core and Basic Region mutant library, and its storage capacity, diversity and randomization is all to meet the requirements of library construction.3. Affinity screening of HIV-1 Tat Core and Basic Region mutant libraryThrough the first part of this study we know that the neutralization epitope of Tat basic region has been preserved. By using two kinds of anti-tat rabbit anti-sera:PET32a-Tat3g-101 and PET32a-Tat1-101, the phage libraries were screened and positive mutant clones were enriched. After two rounds screening we might get the he original sequence of those enriched mutant clones which were stronger affinity, lower trans-membrane activity. Each round after the end of screening,we identified the enriched mutant clones using PCR and sequence analysis.After two rounds screening by using two kinds of anti-tat rabbit anti-sera, The results of PCR identification showed that:①TCBR mutant library inserting fragments from 70% up to 90% after screening, indicating that high affinity mutant fragments are effectively enriched by selection;②from pre-screening to screening, the proportion of the monoclonal containing two basic region fragments in the library is clearly decreased while the proportion of the monoclonal containing single basic region fragment in the library is increased from 55% to 90%. In theory, the monoclonal containing two basic region fragments could easily be obtained through evolutionary selection because of a selective advantage. It seems to suggest that the mutant sites 51X-55X is playing a more important role for containing the neutralization epitope of Tat basic region of large than repeated fragments.We sequenced 40 recombinants after screening two round.The results of sequence analysis showed that:①basic amino acids were enriched at 51 site, which is consistent with theories because natural Tat 51 site is lysine (K);②51 site and 55 site have emerged proline(P). A Japanese experimental results seem to suggest that 51 mutations P are more conducive to penetrating for Tat, which is diametrically opposed to the purpose of this study. Therefore, it requires further study for The same amino acids. One reason maybe that Proline itself seems more easier for stable conformation epitopes.③51S-55P seems to be a meaningful alternative candidate characteristic sequence, which is worthy of further study. Another experimental study in France showed that the Mutant form "STLA Tat" which basic region of 51 lysine (K) site-directed mutagenesis as threonine (T),55 Arg (R) site-directed mutagenesis into a leucine (L), and its' trans-activation activity is eliminated and extracellular toxicity is greatly reduced. According to amino acid classification, both 51 T and amino acids screened characteristic S are polar amino acids, and both 55 L and amino acid screened P characteristic are hydrophobic amino acids, and also with the hydrophobic amino acids. Thus, according to selected characteristics of amino acids and existing research we can view 51S-55P as a meaningful alternative candidate sequence features for further study.In summary, the phage library TCBR has been successfully screened using two kinds of rabbit anti-serum. After sequence analysis, we found 51 site and 55 site of mutation fragments emerging characteristic amino acids.51X for the P\S, and 55X for the Y\P. 51S-55P seems to be a meaningful alternative candidate characteristic sequence.Our study successfully had some results:expressed PET32a-Tat38-61 proteins,and its' neutralization epitope of Tat basic region has been preserved; constructed HIV-1 Tat Core and Basic Region (TCBR) mutant library and screened it using two kinds of rabbit anti-serum; After sequence analysis, we found 51 site and 55site of mutation fragments emerging characteristic amino acids.51X for the P\S, and 55X for the Y\P.51S-55P seems to be a meaningful alternative candidate characteristic sequence. This study attempted to modify HIV-1 Tat Core and Basic Region by making use of molecular evolution based on phage display. We want to get a range of the Candidate mutant sequence which have conformational epitope stability and significantly decreased biological activity. It's a basic research for Tat novel vaccine research and development.