单纯疱疹病毒2型全长糖蛋白D抗原的原核表达及抗原性分析

目的:构建单纯疱疹病毒2型(HSV-2)全长糖蛋白D(gD)基因原核表达质粒,研究其编码的蛋白质在大肠杆菌中的表达,并鉴定重组蛋白的gD抗原性。方法:提取病毒DNA,PCR扩增出gD基因,克隆于原核表达载体pGEX-4T-1,并转化大肠杆菌BL21。PCR、双酶切及测序证实插入的gD基因序列正确后,IPTG诱导表达融合蛋白GST-gD,并进行免疫学鉴定。结果:获得了gDDNA。测序鉴定表明,该序列与Gen Bank中的序列一致,gD基因已正确插入到pGEX-4T-1中。重组融合蛋白表达载体pGEX-4T-gD经IPTG诱导后能在大肠杆菌中高效表达,Western Blotting证实,该蛋白具有天然gD抗原性。结论:成功构建了融合蛋白表达载体pGEX-4T-gD,在大肠杆菌中获得了有效表达,并证实融合蛋白具有gD免疫原性。为进一步研究gD蛋白的免疫学特性,制备gD亚单位疫苗和单克隆抗体奠定了基础。

Prokaryotic expression of full length HSV-2 gD antigen and its antigenicity

Objective To construct the expression plasmid of giutathione stransferase-gD gene (GST-gD) fusion protein, investigate the expression of this protein in Escherichia coli BL21, and identify the antigenicity of the protein. Methods Following extraction of viral DNA, gD gene was amplified by PCR, cloned into the vector pGEX-4T-1, and then transformed into BL21. GST-gD was expressed in BL21 via induction of IPTG and its antigenicity was identified following the exact pGEX-4T-gD was confirmed by PCR, restriction endonuclease digestion, and sequencing. Results Sequencing showed that the sequences of gD gene were identical with those printed in Gene Bank and gD gene was inserted exactly into pGEX-4T-1. GST-gD was highly expressed in Escherichia coli after the recombinant vector was induced by IPTG. Western Blotting confirmed that the recombinant protein had gD immunogenicity. Conclusion The expression vector of GST-gD fusion protein has been constructed successfully and the protein has been expressed effectively in Escherichia coli BL21 and its antigenicity is confirmed, which lays the foundation for further studying the immunological characteristics of gD and for producing gD subunit vaccine and monoclonal antibodies.