Identification and Expression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E ．coil

ZHONG Yan wei; CHENG Jun．SHI Shuang-shuang; el al

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April 2001

ZHONG Yan wei, CHENG Jun．SHI Shuang-shuang, el al, Identification and Expression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E ．coil[J]. Virologica Sinica, 2001, 16(2): 105-108.

Citation: ZHONG Yan wei, CHENG Jun．SHI Shuang-shuang, el al, . Identification and Expression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E ．coil .VIROLOGICA SINICA, 2001, 16(2) : 105-108.

抗HBsAg人源单链可变区抗体的筛选与可溶性抗体的表达

1.
解放军三0二医院传染病研究所基因治疗研究中心．北京100039

摘要

采用噬菌体表面展示技术，以从乙型肝炎病毒(HBV)表面抗原( Ag)阳性血清超速离心纯化的HBsAg为圃相抗原．趴噬菌体单链可变区半合成抗体库中经过5轮“吸附洗脱一扩增筛选过程．获得特异性较强的[-IB~Ag 人源单链可变区抗体(ScFv)克隆并提取质粒，经s I／,NbtI酶切鉴定后．亚克隆到pCANTAB5E表达载体中，转化大肠杆菌XL1一Blue。经IPTG诱导后，表达的可溶性HBsAg特异性ScFv以50％硫酸胺沉淀．经SDS—PAGE电泳表明，XL1．Blue中表达的HBsAg可溶性ScFv的分子量约28 kD。免疫活性检测结果表明，该单链抗体具有鞍强的抗原结合活性和特异性 HmAg人源单链抗体的筛选和表达成功，为今后HBsAg人源抗体的研究和应用奠定厂基础。
- HBsAg
- , 噬菌体
- , 单链抗体
- , 筛选
- , 表达

Identification and Expression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E ．coil

1.
Institute of Infectious Diseasas，The 302 Hospital PLA ，Beifing 100039，China

Abstract

Abstract：Using HBsAg antigen purified from }ⅢsAg positive$ar~Ii1 as the coating antigen，the sin— gle—chain variable fragment(ScFv)antibodv to HBsAg has been screened and idemifed from a semi synthetic phage library by phage display technique The SfiI／NotI scFv DNA fragment was harvested from PHENI—ScFv and inserted into pCANTAB5E．and the recombinant plasmid DNA was used to transf0rm competent host XL1 Blue．After induction with IP，rG fnr 20 hrs，the expressed Ⅲ sAg ScFv in the supernantant of XL1一B1He was precipitated with 50％ ammonium sulfated and demonstrat ed the molecular weight is about 28kDa bv SD PAGE The reactivity and spedficity have be n con— firmed by enzyme—linked irnmuno-sorbent assay f ELISA)．The identification and expression of HB— sAg ScFv from E coli were successfully achieyed
- HBsAg
- , Phage
- , Single chain Fv antibody
- , Screning
- , Expresion

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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

Identification and Expression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E ．coil

1. Institute of Infectious Diseasas，The 302 Hospital PLA ，Beifing 100039，China

Abstract: Abstract：Using HBsAg antigen purified from }ⅢsAg positive$ar~Ii1 as the coating antigen，the sin— gle—chain variable fragment(ScFv)antibodv to HBsAg has been screened and idemifed from a semi synthetic phage library by phage display technique The SfiI／NotI scFv DNA fragment was harvested from PHENI—ScFv and inserted into pCANTAB5E．and the recombinant plasmid DNA was used to transf0rm competent host XL1 Blue．After induction with IP，rG fnr 20 hrs，the expressed Ⅲ sAg ScFv in the supernantant of XL1一B1He was precipitated with 50％ ammonium sulfated and demonstrat ed the molecular weight is about 28kDa bv SD PAGE The reactivity and spedficity have be n con— firmed by enzyme—linked irnmuno-sorbent assay f ELISA)．The identification and expression of HB— sAg ScFv from E coli were successfully achieyed