Fusion Expression And Basic Study On Anti-HIV-1 Ability Of Cell Permeable Peptide Pep-1 And Viral Macrophage Inflammatory Protein-Ⅱ

Objective:Cell Permeable Peptide pep-1 and Viral macrophage inflammatory protein-II (vMIP-II) fusion protein was producted by the prokaryotic expression system, then the functions of penetrate the cell membrane and upregulate many anti-HIV-1 genes were detemined by confocal and quantative real-time PCR, respectively. It will provide basic data to develop vMIP-II into a new and easily-accessible drug to prevent and cure AIDS.Methods:The open reading frame of vMIP-II was obtained by PCR, and the pep-1 was synthesized by chemical method, then both these sequences were cloned into Prokaryotic Expression Vector pET15b. The plasmid pET15b-pep-l-vMIP-Ⅱwas transducted into Escherichia coli BL21 (DE3) plysS. The expression product induced by IPTG was identified and analysed by SDS-PAGE and Western-blot. After purified by Ni-NTA agrose affinity chromatography, it was detected by Laser Scanning Confocal Microscope for observing the function of penetrating cell membrane. In addition, a few major anti-HIVgenes (MX1, MX2, CCL5, APOBEC3G and APOBEC3F) in Jurkat and HMEC-1 cells were determined by qRT-PCR.Results:Sequence analysis showed that the sequence of the prokaryotic expression vector pET15b-pep-l-vMIP-Ⅱwas constructed rightly. Both proteins expressed in the bacterial are major in soluable form. The results by laser confocal microscopy showed that Cell Permeable Peptide pep-1 can carry vmip-II gene through the cell membrane. Quantitative PCR results show that the fusion protein can increase the anti-HIV genes MX1, MX2, CCL5, APOBEC3G and APOBEC3F of Jurkat cells in 3.36,2.21, .40,2.09 and 2.72 times; and increase the anti-HIV genes MX1, MX2, CCL5, APOBEC3G and APOBEC3F of HMEC-1 cells in 57.80,158.63,97.29,2.18and 2.43 times, respectively. Protein vMIP-II can increase the anti-HIV genes MX1, MX2, CCL5, APOBEC3G and APOBEC3F of Jurkat cells in 9.70, 22.03,1.59,1.79 and 1.85 times; and increase the anti-HIV genes MX1, MX2, CCL5, APOBEC3G and APOBEC3F of HMEC-1 cells in 57.77,159.66,108.77,2.36 and 2.31 times, respectively.There were no differences between the two protein.Conclusion:The fusion protein pep-1-vMIP-II was successfully expressed in E. coli, and the fusion protein can penetrate cell membranes into cell nucleus, and thus play a variety function of up-regulation of anti-HIV-1 genes as the protein vMIP-II.