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目的通过基因工程技术,克隆和表达Ⅱ型单纯疱疹病毒gG基因的免疫优势表位片段。方法以PCR法扩增HSV-2的gG基因的免疫优势表位片段;将PCR产物与TOPO定向表达载体连接;然后鉴定含有目的基因的重组表达载体;并转化BL21StarTM菌株诱导表达目的蛋白;以免疫印迹法检测表达的目的蛋白。结果PCR法扩增出616bp的DNA片段;通过PCR法及测序法证实重组质粒中含有616bp的DNA片段,其中99.5%序列与目的基因序列一致。SDS-PAGE结果显示重组蛋白在3h时的表达产量最高。免疫印迹法通过抗gG的单克隆抗体,证实重组蛋白在大肠杆菌中的表达。结论Ⅱ型单纯疱疹病毒的gG免疫优势表位基因片段的成功表达,为制备国产的HSV-2血清诊断试剂提供资料。
Objective To clone and express the immunodominant epitope fragment of herpes simplex virus type 2 gG gene by genetic engineering. Methods The immunodominant epitope fragment of gG gene of HSV-2 was amplified by PCR. The PCR product was ligated to the TOPO-directed expression vector and then the recombinant expression vector containing the target gene was identified. The BL21StarTM strain was transformed to induce expression of the target protein. The target protein was detected by Western blotting. Results The 616bp DNA fragment was amplified by PCR. The 616bp DNA fragment was confirmed by PCR and sequencing, of which 99.5% was consistent with the sequence of the target gene. The result of SDS-PAGE showed that the recombinant protein had the highest yield at 3h. Immunoblotting confirmed the expression of the recombinant protein in E. coli by anti-gG monoclonal antibody. Conclusions The successful expression of gG immunodominant epitope fragment of herpes simplex virus type 2 gene provides information for preparing HSV-2 serum diagnostic reagent.