HSV-1 stocker株糖蛋白G基因膜外区毕赤酵母表达载体的构建及序列分析

目的构建Ⅰ型单纯疱疹病毒型特异性包膜糖蛋白G基因膜外区毕赤酵母表达载体,并进行序列分析。方法PCR扩增HSV-1-gG基因的膜外区,克隆于pGEM-T载体,转化DH5α,提取质粒酶切鉴定后,与pPIC9K载体连接,转化DH5α,筛选阳性克隆,鉴定后转化GS115菌,构建酵母表达载体。对克隆的序列进行分析,预测表达产物的理化特性、抗原性及表达形式。结果获得的重组酵母表达载体pPIC9K-gG,测序结果证实为HSV-1-gG基因,序列分析其高度保守,预测蛋白相对分子质量15870,等电点pI为4.72,包含全部膜外区分值达1.7的6个强抗原决定簇,将以可溶性形式分泌表达于胞外。结论已成功构建HSV-1stocker株糖蛋白G基因膜外区毕赤酵母表达载体。

Construction and Sequencing of Recombinant Pichia pastoris Expressing Glycoprotein G Ectodomain Gene of Herpes Simplex Virus Type 1 Stocker Strain

Objective To construct the Pichia pastoris expression vector of the glycoprotein G(gG) ectodomain gene of herpes simplex virus type 1(HSV-1) stocker strain and analyze its sequence.Methods Amplify the ectodomain of HSV-1 gG gene by PCR and clone into vector pGEM-T,then transform to competent E.coli DH5α.Extract recombinant plasmid pGEM-T-gG1 by alkaline lysis and ligate to expression vector pPIC9K,then transform to E.coli DH5α.Screen recombinant plasmid pPIC9K-gG1,analyze its sequence and predict the physiochemical property,antigenicity and form of expressed product.Results Recombinant yeast expression vector pPIC9K-gG was constructed.Sequencing result proved that the cloned fragment was highly conserved ectodomain of HSV-1-gB gene.By the prediction using bio-software,the expressed product contain the six strong antigenic determinants of the whole ectodomain and existed in a form of soluble protein,and its relative molecular weight and isoelectric point were 15 870 and 4.72 respectively.Conclusion The Pichia pastoris expression vector of ectodomain of gG gene of HSV-1 stocker strain was successfully constructed.