Construction of prokaryotic expression vector carrying HIV Tat gene and the expression,purification and identification of recombinant Tat in E. coli

Objective: To construct the prokaryotic expression vector carrying HIV-1 Tat gene and to purify the fusion protein in E.coli.Methods: The DNA fragment of the Tat gene from pcDNA3.1(+) / Tat101 was cloned into the prokaryotic expression vector pET-28a(+),named pET-28a(+)-Tat.Then the recombinant vector was transformed into E.coli BL21(DE3).The expression of the histidine-tagged(His-Tag) fusion protein was induced with isopropyl-β-D-thiogalactopyranoside(IPTG).The fusion protein was detected and purified by Western blot and immobilized Ni2 + absorption chromatographic column,respectively.Results: The prokaryotic expression vector carrying Tat gene was successfully constructed.The expression of the His-Tag fusion protein in E.coli BL21(DE3) was detectable.Finally,the fusion protein with the relative molecular weight of 18 × 103 was gained after purified using the affinity chromatographic column.Conclusion: Recombinant Tat gene can be expressed in E.coli BL21(DE3) and the fusion protein obtained can be functionally active.