Expression Of HIV-1 Gp-41 Recombinant Antigen In Vivo And In Vitro Of Escherichia Coli And Analysis Of Its Immunoreactity

In recent years, HIV has shown a rapid epidemic trend worldwide. As the main method of HIV initial diagnosis at present, HIV antibody diagnostic kit has enormous market value. With the development of biotechnology, the preparation of HIV-1 gp41 recombinant antigen materials which is of favorable immunoreactivity by the means of genetic engineering, has profound significance to the research and development of the HIV antibody diagnostic kit.Primarily, this article synthesized the plasmid template pET-28a(+)-gp41 which contained codon-opitimized gp41 gene. Then, the gene sequence including gp41’s main epitopes and three important domains (NHR, CHR, MPER) was amplified and obtained by PCR. And by fusing with hydrophilic chaperone DsbA, the gp41 truncated fragment was successfully expressed in vivo of E. coli. In the optimal expression conditions (37℃,0.2 mM IPTG, culturing 10 h after inducing), the yield of gp41 fusion protein was 8.7 μg/mL.Due to the low expression level of gp41 fusion protein in vivo of E. coli, which couldn’t meet the requirement for the preparation of gp41 antigen, so this article took advantage of the cell-free system to express gp41 in vitro of E. coli. Firstly, cell-free expression plasmid pIVEX2.4c-gp41 was successfully constructed after cloning the full-length gp41 gene by PCR. And the full-length gp41 protein was achieved in cell-free expression system. The effects of expression of soluble gp41 by P-CF mode and D-CF mode in cell-free system were further investigated. And results showed that all detergents couldn’t effectively resuspend and dissolve gp41, while in the optimal conditions of D-CF mode (0.2% final concentration of Brij78), the expression level of soluble gp41 was 650 μg/mL.Soluble gp41 recombinant protein was expressed by D-CF mode in cell-free system. After purification, concentration and replacing buffer, the gp41 recombinant protein was obtained with a purity of 95.6%, a concentration of 1.5 mg/mL and a recovery rate of 70%. Then, latex test strips were prepared by using gp41 recombinant protein obtained in this article to detected various samples (3 HIV-1 positive samples with different antibody titre,3 HIV-1 negative samples,1 TP positive sample and 1 HBeAb positive sample). Results preliminarily proved that gp41 recombinant protein prepared in this article had favorable immunoreactivity.This article is committed to the research on recombinant expression of HIV-1 gp41 in vivo of E. coli and in cell-free system in vitro of E. coli. By optimizing the cell-free expression system, full length gp41 was solubly expressed. After affinity purification, the target protein showed favorable immunoreactivity through the assessment of preparing latex method strips. In general, this study provides a gp41 recombinant antigen with potential value, which has profound significance for the research and development of the HIV antibody diagnostic reagents.