Clone of HIV-1 p24 gene and the pilot study of its toxicity to Chinese hamster ovary cells

Abstract

Objective To express HIV-1 P24 in Escherichia coli (E.coli) and Chinese hamster ovary (CHO) cells and observe toxicity of P24 protein to CHO cells in vitro.Methods The p24 gene extracted from the HIV-1 patient's peripheral blood was amplified by RT-PCR.Then p24 gene was inserted into pEGFP-C2 after digested by Hind Ⅲ and EcoR Ⅰ,subsequently the recombinant plasmid was transformed to CHO cells,expression of green fluorescent protein (GFP) was observed.Meanwhile,p24 gene was inserted into pET24a after digested by HindⅢ and Xho Ⅰ,then the recombinant plasmid was transformed to E.coli BL21 (DE3),and highly expressed after IPTG induction.The correct insert in the recombinant plasmid was confirmed by double enzymedigestion and SDS-PAGE electrophoresis.Western blot and ELISA were used to analyze purity and antigenicity of the expression product.Purified recombinant P24 protein was added to the CHO cell culture supematant,and toxicity of P24 to CHO cells was observed.Results Two hours after P24 transformation to CHO cells,the expression level of GFP was the highest and the concentration of recombinant P24 protein was 15 ng/L.Then GFP started to decrease gradually and disappeared 8 hours after transformation.And death of all the CHO cells was observed.The p24 was highly expressed in E.coli BL21.CHO cells died gradually after recombinant P24 protein added into the culture supematant of CHO cells.Conclusions pEGFP-C2-p24 is constructed as an expression vector of HIV-1 p24 and could express transiently in CHO cells.pET24a-p24 is also constructed and could highly express in E.coli.The expression product has good antigenicity.The recombinant P24 protein,which is toxic to CHO cells,may be involved in nosogenesis of HIV-1. Key words: HIV-1; P24 protein; Gene express