新型抗HIV-1蛋白GRFT的构建、表达及活性研究

根据已知的新型抗HIV-1蛋白Griffithsin(GRFT)基因氨基酸序列,推测其DNA编码序列,对密码子优化及修饰后进行全基因化学合成,连接到原核表达载体pET28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达,获得目的蛋白.SDS-PAGE和Western Blot分析结果表明,目的蛋白得到良好表...

Construction, Expression and Activities of a Novel Anti-HIV-1 Protein GRFT

A DNA sequence encoding GRFT was synthesized according to the deduced amino acid sequence of GRFT, using an E. coli codon preference table, and cloned into the prokaryotic expression vector pET28a (+). Then the recombinant plasmid was transfered into E.coli BL21(DE3). The target protein was obtained after induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The results of SDS-PAGE and Western Blot showed that the protein was well expressed and had antigenic activities. When different parameters were optimized, the highest production could account for 55.84% of total bacterial protein. Gel scan analysis showed that the purity was 94.06% after the target protein refolding and purification with Ni2+-NTA affinity chromatography. The antigenic and cellular biding activities of target protein were identified by Dot-ELISA and IFA methods based on the HIV-1 infection target cell models prepared by our laboratory. The results suggested that the recombinant protein has special recognition and binding activity with target cells. This research will settle solid foundation for further investigate of anti-HIV-1 genetic engineering agents and targeted therapy.