Expression of Soluble Human SeFv Against Envelope Protein E2 of 
Hepatitis C Virus Antigen in E．coli

CHENG Jun; Shi Shuang—shuang．ZHONG Yan—wei; XIA Xiao—bingWANG Gang; WANG Lin; LIU Yan; CHEN Ju-mei

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June 2001

CHENG Jun, Shi Shuang—shuang．ZHONG Yan—wei, XIA Xiao—bingWANG Gang, WANG Lin, LIU Yan, CHEN Ju-mei, Expression of Soluble Human SeFv Against Envelope Protein E2 of Hepatitis C Virus Antigen in E．coli[J]. Virologica Sinica, 2001, 16(3): 220-223.

Citation: CHENG Jun, Shi Shuang—shuang．ZHONG Yan—wei, XIA Xiao—bingWANG Gang, WANG Lin, LIU Yan, CHEN Ju-mei, . Expression of Soluble Human SeFv Against Envelope Protein E2 of Hepatitis C Virus Antigen in E．coli .VIROLOGICA SINICA, 2001, 16(3) : 220-223.

丙型肝炎病毒包膜蛋白E2可溶性单链可变区抗体在大肠杆菌中的表达

1.
解放军第三0二医院传染府研究所基因治疗研究中心，北京100039

摘要

利用分子生物学技术，构建表达丙型肝炎病毒(HCV)包膜蛋白卫的人源单链可变区抗体(scFv)的原核表达载体，并在太肠杆菌JM109中表达可溶性的HCV-E2一scFv。以重组的HCV E2蛋白为包被抗原，利用噬苗体抗体库的表面展示技术，筛选到含有HCV-E2-ScFv基因的噬苗体克隆．从噬菌体抗体阳性克隆中提取质粒，经^lm 】／￡I 酶切鉴定后，该SeFv基因由750bp组成，将其亚克隆到~ ITAB5E载体中，转化太肠杆菌．UMI~，提取质粒进行 DNA序列测定，符合ScFv的基因结构特点。IPIG诱导转化的大脑杆苗JM109，在其培养上清中获得了可溶性HCV 单链可变区抗体的表达。酶联免疫吸附法(ⅡJsA)证实表达的HCV-E2一ScFv具有与重组HCV E2蛋白的反应活性和特异性，对转化的J／vll09大脑杆菌上清中表述的HCV．E2-ScFv进行聚丙烯酰胺凝胶电泳(PAGE)，证实表达的 HCV-E2-sc 的分子量为28kn。为应用HCV．E2．ScFv进行肝组织免疫组织化学和细胞内免疫基因治疗研究奠定了基础。
- 丙型肝炎病毒
- , 包膜蛋白
- , 单链可变区抗体
- , 表达

Expression of Soluble Human SeFv Against Envelope Protein E2 of Hepatitis C Virus Antigen in E．coli

1.
Gene Theropy Research Center,Institute of infectious,the 302 hospital of PLA,Beijing 100039,China

Abstract

To construct expressive vector for human ScFv against E2 protein of hepatitis C virus (HCV-E2-ScFv),and express soluble HCV-E2-ScFv in E.coli JM109,using phage display technique,the recombinant phages were panned by recombinant E2 antigen which was coated in a microtiter plate,after five rounds of biopanning,46 clones were identified specific to E2 antigen.750 bp fragment could be released from the plasmid of positive phage colones,and the sequenle analysis indicated that we have got the ScFv BNA fragment.And then DNA fragment was inserted into expressive vector pCANTAB5E and E.coli host JM109 was transformed and induced by IPTG.The specificity of ScFv in the culture medium was evaluated by enzyme-linked immunosorbent assay(ELISA).The molecular weight of expressed HCV-E2-ScFv is 28kD by SDS-polyacrymide gel electrophoresis(PAGE).The expressed HCV-E2-ScFv will be useful in the immunohistochemical study of liver tissue and gene therapy against HCV infectiion.
- HCV
- , E2
- , Single chain Fv antibody
- , Expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Expression of Soluble Human SeFv Against Envelope Protein E2 of Hepatitis C Virus Antigen in E．coli

1. Gene Theropy Research Center,Institute of infectious,the 302 hospital of PLA,Beijing 100039,China

Abstract: To construct expressive vector for human ScFv against E2 protein of hepatitis C virus (HCV-E2-ScFv),and express soluble HCV-E2-ScFv in E.coli JM109,using phage display technique,the recombinant phages were panned by recombinant E2 antigen which was coated in a microtiter plate,after five rounds of biopanning,46 clones were identified specific to E2 antigen.750 bp fragment could be released from the plasmid of positive phage colones,and the sequenle analysis indicated that we have got the ScFv BNA fragment.And then DNA fragment was inserted into expressive vector pCANTAB5E and E.coli host JM109 was transformed and induced by IPTG.The specificity of ScFv in the culture medium was evaluated by enzyme-linked immunosorbent assay(ELISA).The molecular weight of expressed HCV-E2-ScFv is 28kD by SDS-polyacrymide gel electrophoresis(PAGE).The expressed HCV-E2-ScFv will be useful in the immunohistochemical study of liver tissue and gene therapy against HCV infectiion.