Ⅰ型单纯疱疹病毒gD基因片段的高效表达及抗原性分析

High Efficiency Expression of the Gene From Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D

目的:获得高表达的I型单纯疱疹病毒(HSV)被膜糖蛋白gD(简称gD1)基因的工程菌。方法:通过计算机分析,筛选出疱疹病毒gD1中优势抗原决定簇的基因片段。将克隆的基因片段插入表达载体pTrxA内,转化大肠杆菌Rosetta,以异丙基βD硫代半乳糖苷诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)分析表达产物。结果:PCR扩增出约930bp的gD1编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变。所构建pTrxAgD1重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致。含有pTrxAgD1重组质粒的大肠杆菌Rosetta诱导后得到了高效表达,SDSPAGE显示表达产物约48kDa。免疫印迹结果表明表达产物具有较好的抗原性。结论:成功构建了pTrxAgD1表达质粒,实现了成熟gD1蛋白在大肠杆菌中的高效表达,表达产物具有好的抗原性。

Objective ： To obtain the high expression of Herpes Simplex Virus Type 1 （ HSV-1 ） Glycoprotein D gene. Methods： The Herpes Simplex Virus Type 1 （HSV-1）Glycoprotein D （gD1） gene fragment containing dominant antigen epitopes confirmed by computer analysis was cloned by PCR technical and inserted into plasmid vector pTrxA. Then the recombinant plasmid was transformed into Rosetta. The expressed product was analyze by SDS-PAGE. Results ：930 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100 % homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about 48kDa, Western blotting indicated that the antigenicity of the protein was good. Conclusion： The plasmid pTrxA-gdl was constructed and a high efficiency expression of the gdl gene from Herpes Simplex Virus Type 1 （HSV-1）strain was made. The expressed product shows a good antigenicity.