Construction, prokaryotic expression and immunogenic analysis of HXB2 subtype Tat mutant of human immunodeficiency virus type-1 / 中华传染病杂志

ABSTRACT

Objective To construct shifting mutant of cysteine-rich region to 3?@terminal of Tat gene of human immunodeficiency virus type-1 (HIV-1) HXB2 strain, and to analyze the immunogenicity of mutant protein (Tat-cct) after prokaryotically expressed and purified. Methods The cysteine-rich region (nucleotides 64--111) of Tat gene was shifted to 3'terminal of Tat of HIV-1 HXB2 strain by polymerase chain reaction (PCR) and Tat mutant DNA sequence was obtained. Prokaryotie express plasmid pET32a-Tat-cct was constructed and transformed into E. coli BL21 (DE3), then Tat-cct protein was expressed and purified. BALB/c mice were immunized with the fusion protein Tat-cct, and immunogenicity of the immunized serum was detected by enzyme-linked immunosorbent assay (ELISA). Results The recombinant plasmid pET32a-Tat-cct expressed in E. coli BL21 (DE3) and the relative molecular mass of the purified fusion protein was 31 000. The serum antibody titer of mice immunized with Tat-cct recombinant protein was 1 1600, which binded specifically with both Tat-ect protein and Tat protein (amino acids 1-101). Conclusions The recombinant protein Tat-cct of Tat mutant strain can be expressed efficiently in E. coli and well retains immunogenicity, which provides valuable information for basic research of HIV-1 Tat vaccine.