Prokaryotic Expression and Immunoreactivity of HIV-1 HXB2 Tat Core and Basic Region Polypeptide Tat_(38-61)

Objective To construct a prokaryotic expression vector for HIV-1 Tat core and basic region polypeptide Tat38-61,express fusion protein in E.coli,identify the expressed product and analyze its immunoreactivity.Methods The gene encoding Tat38-61 was amplified by PCR from HIV-1 HXB2 Tat1-101 gene and cloned into prokaryotic expression vector pET32a(+).The constructed recombinant plasmid pET32a(+)-Tat38-61 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by Ni2+-NTA column affinity chromatography and determined for immunoreactivity by ELISA.Results Both restriction analysis and sequencing proved that recombinant plasmid pET32a(+)-Tat38-61 was constructed correctly.SDS-PAGE showed a target protein band with relative molecular mass of about 21 300.The expressed product,mainly in a soluble form,contained 67.4% of total somatic protein and reached a purity of more than 97% after purification.ELISA showed specific reactions of the fusion protein with both rabbit anti-PEPTIDE-Tat1-101 serum and HIV-positive serum.Conclusion The prokaryotic expression vector for HIV-1 Tat core and basic region polypeptide Tat38-61 was successfully constructed,and Tat38-61 fusion protein was expressed and purified.The basic area epitope of the fusion protein was remained well,which laid a foundation of construction and screening of Tat38-61 mutant phage display library.