Construction Expression And Purification Of Mutant Of Human Immunodeficiency Virus Type-1 Tat

In the post decades, infection of HIV mushroomed in the world, nowdays is a serious disease threaten the health of human beings. It is an important method to develop the effective anti-HIV vaccine to lower the morbility rate of infectors and avoid more people to catch HIV.HIV is classified into type 1 and type 2, the HIV-1 is widespread now. HIV-1 Tat (trans-activator of transcription) protein is a key viral regulatory protein and is necessary for viral gene replication, spreading and pathopoiesis.Integrity Tat protein has six fuctions region including N-terminal region, cysteine richregion, core region, basic region gln-rich segment and C-terminal regions. Antibodies toward HIV-1 Tat, which are inversely correlated to disease progression in the infection of HIV, may cause the HIV-1 blood serum masculine infected person for a lack of disease progression for a long time. The Tat protein induces the neutralizing antibody can suppresses the production of duplication and pathopoiesis in vitro experiment and animal.In HIV Tat vaccine's research, the lab establishes PET32a-Tat(△C) protein which deletes the cystein-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its expression level and stability in E.coli and remain good immunogenicity in our laboratory, but it produces antibody titer is lower after immunity mouse.It is highly effective to obtain a new immunity by reconstruct to the Tat 's primary structure.It is important significance to develop the prophylactic and therapeutic Tat vaccine.Three parts:1. Construction of mutant of HIV-1 HXB2 Subtype Tat shifted the cysteine-rich region to C terminal and its prokaryotic expression and immunogenicity analyses Tat C terminal is important epi-position antigen to express cellular immunity. The cysteine-rich region has 7 conservative cysteine (C22, C25, C27, C30, C31, C34 and C37), has many kinds of extracellular functions and adjuvant function.Tat mutant DNA shifted the cysteine-rich region (64-111 nucleotides, 22-37 amino acids) to 3′-terminal of Tat, named as Tat-cct DNA, was obtained by PCR and cloned into pET32a vector. pET32a -Tat-cct plasmid was established and transformed into E.coli BL21 (DE3) strains to express protein, and the relative molecular mass of the protein was 31 000,and purifing the expressed product by the Ni-NTA column and the dense of the purified PET32a-Tat(1-101aa) protein was 3.46g/L. BALB/c mice were immunized with the fusion protein, and titer of the serum from mice immunized with pET32a-Tat-cct protein was 1:1600, and the antibody can react specifically with pET32a-Tat -cct protein and pET32a-Tat protein (1~101 aa).2. Construction of mutant of HIV-1 HXB2 Subtype Tat shifted the cysteine-rich region to N terminal and its prokaryotic expression and immunogenicity analysesTat N terminal is important epi-position antigen to express cellular immunity. Tat mutant DNA shifted the cysteine-rich region (64-111 nucleotides, 22-37 amino acids) to N-terminal of Tat deleting the cysteine-rich region Tat(△C), named as Tat (CN) DNA, was obtained by PCR and cloned into pET32a vector. PET32a -Tat (CN) plasmid was established and transformed into E.coli BL21 (DE3) strains to express protein, and the relative molecular mass of the protein was 31000,and purifing the expressed product by the Ni-NTA column and the dense of the purified pET32a -Tat (CN) protein was 5.17 g/L. The expressed and purified pET32a-Tat (CN) protein was analyzed by SDS-PAGE, then testify the reactivity of the purified protein by indirect ELISA and Western-blot. Indirect ELISA and Western-blot showed that anti-sera of pET32a-Tat protein from rabbit could react specifically with pET32a-Tat (CN) protein3. Construction of HIV-1 HXB2 Subtype PR recombination protein and its prokaryotic effective expressionHIV Tat cysteine-rich region has special structure and adjuvant function, PR is a necessary enzyme to duplicate HIV. PR recombination DNA shifted the HIV-1 Tat cysteine-rich region (64-111 nucleotides, 22-37 amino acids) to 3′-terminal of PR, named as PR (CC) DNA, was obtained by PCR and cloned into pET32a vector. pET32a - PR (CC) plasmid was established and transformed into E.coli BL21 (DE3) strains to express and purify fusion protein pET32a- PR (CC).the dense of the purified fusion protein pET32a-PR (CC) was 4.32 g / L by SDS-PAGE, and the relative molecular mass of the protein was 31600. The results possibly provide a new immunity original for further development of HIV-1 Tat vaccine.We have deep research to the HIV-1 Tat cysteine-rich region from crosswise and longitudinal.The mutants of HIV-1 HXB2 subtype Tat which shifted the cysteine-rich region to C terminal and N terminal are successfully established and expressed, and remain good antigenicity with the anti-sera of pET32a-Tat protein from rabbit,and provide good fundament for further development of their antibodies from rabbit.The recombinant protein of pET32a - PR (CC) was successfully established into E.coli BL21 (DE3) strains, not only it provides a kind of new pattern for structure and function's research of Tat protein, but also it provides a new immunity original for further development of HIV-1 Tat vaccine.