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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。
OBJECTIVE: To construct the prokaryotic expression plasmid of herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD) gene and study the expression of its encoded protein in E. coli and to identify the gD antigenicity of the recombinant protein. Methods: The gD gene was extracted by PCR and cloned into prokaryotic expression vector pGEX-4T-1 and transformed into E. coli BL21. After PCR, double enzyme digestion and sequencing confirmed that the sequence of inserted gD gene was correct, IPTG induced the expression of fusion protein GST-gD, and immunological identification. Results: gDNA was obtained. Sequence analysis showed that the sequence was consistent with the sequence in Gen Bank, and the gD gene was correctly inserted into pGEX-4T-1. Recombinant fusion protein expression vector pGEX-4T-gD induced by IPTG in E. coli expression, Western Blotting confirmed that the protein has the natural gD antigen. CONCLUSION: The fusion protein expression vector pGEX-4T-gD was successfully constructed and was expressed efficiently in E. coli. The fusion protein was confirmed to have gD immunogenicity. To further study the immunological properties of gD protein, preparation of gD subunit vaccine and monoclonal antibody laid the foundation.