Construction Of Phage Libraries Displaying HIV-1 Tat38-61(51N/55N) Basic Region Mutation And Affinity Screening

Partâ… Construction of Phage Libraries Displaying HIV-1 Tat38-61(51N/55N) Basic Region Mutation LibrariesObjective To construct a phage-displayed random combinatorial library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. Methods Here, we successfully introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR using primers containing the random nucleotide sequences, and used the randomly mutated full-length Tat as templates to generate Tat38-61(51N/55N) mutants. We introduced the Xba I recognition sequences at 5′and 3′terminals by PCR with designed primers, then cloned the Tat38-61(51N/55N) mutants into Xba I site in the phagemid vector pCANTAB5S, and transformed the recombinants into E. coli TG1. We constructed the combinatorial phage library displaying HIV-1 Tat38-61 with random mutation at the sites of 51 and 55 amino acids in basic region by the rescue of help virus M13KO7 from the transformed E. coli TG1. Results Our results showed that this library consisted of about 5.0×10⁶ colonies with the titer of 2.65×10¹² TU/ml. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. Conclusion These results demonstrated that the constructed phage library could meet the requirements for the following molecular evolution screening which could produce the Tat mutants for the development of new Tat vaccine candidates. Partâ…¡Affinity Screening of HIV-1 Tat38-61(51N/55N) Basic Region Mutation LibrariesObjective For affinity screened the libraries using anti-Tat38-61 rabbit antibody,anti-Tat38-101 rabbit antibody,anti-Tat38-101 rabbit serum in order to obtain the original sequence of novel immunogen. Methods We used anti-Tat38-61 rabbit antibody,anti-Tat38-101 rabbit antibody,anti-Tat38-101 rabbit serum to screen 5 rounds the HIV-1 Tat38-61(51N/55N) Basic Region Mutation Libraries, then choosed randomly 10 positive colonies in the third,fourth and fifth round to proceed sequence analysis every screening. Results This screening experiment was effective. The process of F0-F5 screening used anti-Tat38-61 rabbit antibody, the change of 5S was: 10 to 4 to 3 to 4 to 3 to 4; The process of F0-F5 screening used anti-Tat38-101 rabbit antibody,the change of 5S was: 10 to 7 to 3 to 0 to 0 to 0; The process of F0-F5 screening used anti-Tat38-61 rabbit antibody, the change of 5S was: 10 to 4 to 1 to 0 to 0 to 0; All of 5S presented gradually reduce and steady. The validity screening experiment was assured. We obtained several repeat sequences in every screening libraries and common sequences in libraries. In all sequences colonies of three screening experiment, 51P-55L( 4 repeat),51Q-55L( 3 repeat) had obtained in positive sequences,and so on. Conclusion we got some relevance 51 and 55 amino acids mutants in our screening experiment, and obtained some typical repeat sequence mutants, which can be used to develop new Tat vaccine candidates.Partâ…¢Construction and expression of HIV-1 Tat38-61 (51N/55N) mutant proteinsObjective To construct HIV-1 Tat38-61(51N/55N) mutants fusion proteins, and tested the immunogenicity, then found the different between the mutants proteins and nature proteins. Methods We choosed three typical sequence through affinity screening, anti-Tat38-61 rabbit antibody, anti-Tat38-101 rabbit antibody, anti-Tat38-61 rabbit serum, the primers were designed according to the Tat sequence of HIV-1 strain and the preferred codons of E.coli. The 51 and 55 amino acid random mutated HIV-1 full-length Tat, and then obtained Tat38-61 encoded sequence for template by PCR, as a result, we obtained the Tat38-61 encoded sequence which mutated randomly the 51 and 55 amino acid successfully, and sequenced after inserted into pMD18-T vector by T/A cloning. Then the correct Tat38-61 was inserted into pET32a vector to construct prokaryotic expression plasmids pET32a-Tat38-61 and the recombinant plasmid was transformed into E.coli BL21(DE3) for expression . The fusion proteins Tat38-61 was expressed by induction of IPTG, then purified by Ni-NTA column. Results We amplificated the DNA fragment of Tat38-61 with 72bp, and the construction of recombinants expression plasmid pET32a-Tat38-61 were succeeded by restriction enzyme cutting site analysis and sequence identification, and SDS-PAGE showed that we obtained pET32a-Tat38-61 with 21300D in E. coli TG1, and testified by competition restraint experiment with nature pET32aa-Tat 38-61 and pET32aa -TatE fusion proteins to find the difference between mutant and nature protein and analysis the change of mutant biology activity. Conclusion Tat38-61 epitope was the main epitope of Tat, nature Tat38-61-pET32a and TatE-pET32a had the best suppressive effect against with nature Tat38-61 antibody. Three mutant protein had suppressive effect against with nature Tat38-61 antibody, but the effect was less than nature Tat38-61-pET32a and TatE-pET32a,it was accord with reality.