Construction and Expression of Prokaryotic Expression Vector for tat (B41-101N)Fusion Gene of HIV-1 HXB2 Strain

Objective To construct the prokaryotic expression vector for tat(B41-101N)fusion gene of HIV-1 HXB2 strain, express and purify Tat (B41-101N)fusion protein and lay a foundation of study on its immunogenicity. Methods Tat41-101 amino acids(aa)were added to the N-terminus of Tat protein of native HIV-1 HXB2 strain. The tat41-101 and tat1-101 gene fragments, encoding Tat41-101aa and Tat1-101aa respectively, were amplified by PCR and used to generate fusion gene tat(B41-101N)by splic-ing by overlap extension PCR(SOE PCR). Prokaryotic expression vector pET32a-tat(B41-101N)was constructed and identified by restriction analysis and sequencing, then transformed to E. coli BL21 (DE3)for expression under induction of IPTG. The expressed fusion protein Tat (B41-101N)was purified by Ni2+-NTA column affinity chromatography and identified by SDS-PAGE, Western blot and ELISA. Results The fusion gene ta(tB41-101N)at a length of about 500 bp was amplified by SOE PCR. Recombinant plasmid pET32a-tat (B41-101N)was proved to be constructed correctly by both restriction analysis and sequencing. SDS-PAGE showed that Tat(B41-101N)fusion protein, with a relative molecular mass of about 36 000, was expressed, which contained about 7% of total so-matic protein and reached a purity of about 60% after purification. Ta(tB41-101N)fusion protein showed specific reaction with mouse McAb against Tat and anti-HIV positive sera, as proved by Western blot and ELISA. Conclusion The prokaryotic expression vector for tat (B41-101N)fusion gene of HIV-1 HXB2 strain was successfully constructed, and the fusion protein with immunoreactivity of native Tat protein was expressed, which laid a foundation of further study on novel HIV Tat vaccine.