单纯疱疹病毒包膜糖蛋白gC合成及验证  

Synthesis and verification of herpes simplex virus envelope glycoprotein gC

作　　者：张宇[1] 姚煦[1] 顾汉艳[1] 王宝玺[1] 刘军[2] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所,南京210042 [2]南京大学医学院附属鼓楼医院皮肤科

出　　处：《中华皮肤科杂志》2014年第8期578-582,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81171501）;江苏省自然科学基金（BK2011127）

摘　　要：目的 利用基因工程技术表达单纯疱疹病毒（HSV）包膜糖蛋白（gC）并进行验证.方法 分两段合成gC蛋白,分别合成基因GC-F和GC-R,并分别将基因亚克隆进pSumo-Mut（含Stu Ⅰ和Xho Ⅰ酶切位点）和pCzn1（含Nde Ⅰ和XhoI酶切位点）表达载体,获得pSumo-Mut-GC-F和pCzn 1-GC-R质粒.将两重组质粒转化ArcticExpress^TM（DE3）原核宿主菌,异丙基硫代半乳糖苷（IPTG）诱导表达目的蛋白,经Ni柱亲和纯化,最终获得高纯度蛋白.采用Western印迹法检测重组GC-F和GC-R两段蛋白与HSV IgG的结合情况.结果 应用全基因合成方法获得GC-F和GC-R基因片段,经SDS-PAGE鉴定分析,主要分布于沉淀层,经亲和层析后获得的蛋白纯度在80％以上,并可被人抗HSV-1抗体（IgG）阳性血清识别.结论 利用基因工程技术构建融合表达载体,并经IPTG诱导成功表达gC蛋白,与HSV-1感染血清反应阳性,是后续进行功能研究的理想实验材料.Objective To synthesize herpes simplex virus （HSV） envelope glycoprotein gC using gene engineering techniques,and to verify its expression.Methods Two separate parts of the HSV envelope glycoprotein gC,i.e.,GC-F and GC-R,were respectively synthesized.The GC-F and GC-R genes were synthesized,subcloned into the expression vectors pSumo-Mut （containing recognition sequences for endonucleases Stu1 and XhoI） and pCzn1 （containing recognition sequences for endonucleases NdeI and XhoI） respectively to form the recombinant plasmids pSumo-Mut-GC-F and pCzn1-GC-R.E.coli BL21 Arctic Express （DE3） cells were transformed with the two recombinant plasmids separately.Isopropyl thiogalactoside （IPTG） was used to induce the expression of target protein which was subsequently purified by nickel affinity chromatography.Finally,Western blot was performed to verify the reactivity of the synthesized protein with the sera of HSV-1-positive patients.Results Both GC-F and GC-R genes were synthesized by a total gene synthesis method.As sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） showed,the fusion proteins were mainly distributed in the sediment layer.The purity of GC-F and GC-R proteins was over 80％ after purification by affinity chromatography.Western blot showed that both of the proteins were reactive with anti-HSV-1 antibody-positive sera.Conclusions Fusion expression vectors have been constructed for the gC protein,and IPTG successfully induces its expression.Moreover,the resulting proteins could react with anti-HSV-1 antibody-positive sera,and may serve as an ideal experimental material for next functional study.

关 键 词：湿疹 单纯疱疹病毒属 包膜糖蛋白 

分 类 号：R752.11[医药卫生—皮肤病学与性病学]