Ⅰ型单纯疱疹病毒gG基因片段的高效表达、纯化

High expression of HSV-1 glycoprotein G gene fragment and purification of the expressed protein

目的 获得具有优势抗原表位的Ⅰ型单纯疱疹病毒糖蛋白G的表达抗原并纯化鉴定。方法 用PCR技术扩增经分析筛选出的HSV1 gG蛋白中优势抗原决定簇较集中的基因片段。将该段基因克隆至PGEX 4T 2表达载体内 ,转化大肠杆菌TGl,对重组体进行诱导表达 ,表达产物主要以可溶性形式存在 ,使用硫酸铵沉淀后采用Sepharose阴离子交换层析进行纯化。纯化的蛋白抗原包被酶联板分别对抗HSV1 IgM阳性血清和正常人血清标本进行检测。 结果 在原核表达载体中成功地克隆了HSV1 gG ,电泳分析表明在相对分子质量 4 3 5kD处有HSV1 gG/GST融合蛋白的高效表达 ,并纯化获得了纯度达 90 %的表达蛋白。ELISA分析初步证实表达产物具有较好的抗原性和特异性。结论 成功地克隆、表达了高纯度的具有良好免疫原性的HSV1 gG蛋白 ,为研制高质量的HSV

Objective To express the antigen of herpes simplex virus type 1 glycoprotein G with dominant antigenic determinants,and purify and identify the expressed protein.Methods The herpes simplex virus type 1 glycoprotein G gene fragment containing dominant antigenic determinants was amplified by PCR and cloned into the expressive vector PGEX-4T-2.The recombinant was induced by IPTG to express target protein,which exists in the form of soluble protein analysed by SDS-PAGE.The expressed product was purified by DEAE-Sepharose FF anion exchange chromatography following ammonium sulfate fractionation step,and then analysed by ELISA test.Results The plasmid PGEX-4T-2/HSV1-gG was reconstructed and expressed a Mr 43 5kD gG1/GST fusion protein in E.coli,which reached a purity of 90% by purification and showed good antigenicity and specificity by ELISA test.Conclusion HSV1-gG gene fragment with good immunogenicity was successfully expressed.This laid a foundation of developing good reagents for HSV-1 immunoassay.