抗HBsAg人源单链可变区抗体的筛选与可溶性抗体的表达 |
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引用本文: | 钟彦伟,成军,施双双,夏小兵,李克,董菁,刘妍,杨继珍. 抗HBsAg人源单链可变区抗体的筛选与可溶性抗体的表达[J]. 中国病毒学, 2001, 16(2): 105-108 |
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作者姓名: | 钟彦伟 成军 施双双 夏小兵 李克 董菁 刘妍 杨继珍 |
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作者单位: | 解放军三○二医院传染病研究所基因治疗研究中心, |
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基金项目: | 国家自然科学基金资助(39900130) |
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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(HBsAg)阳性血清超速离心纯化的HBsAg为固相抗原,从噬菌体单链可变区半合成抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得特异性较强的HBsAg人源单链可变区抗体(ScFv)克隆并提取质粒,经SfiⅠ/NotⅠ酶切鉴定后,亚克隆到pCANTAB5E表达载体中,转化大肠杆菌XL1-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,该单链抗体具有较强的抗原结合活性和特异性。HBsAg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了基础。
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关 键 词: | HBsAg;噬菌体;单链抗体;筛选;表达 |
文章编号: | 1003-5125(2001)02-0105-04 |
修稿时间: | 2000-03-23 |
Identification andExpression of Human ScFv Against Surface Antigen of Hepatitis B Virus in E.coli |
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Abstract: | Using HBsAg antigen purified from HBsAg positive serum as the coating antigen, the single-chain variable fragment (ScFv) antibody to HBsAg has been screened and identified from a semi-synthetic phage library by phage display technique. The SfiI/NotI ScFv DNA fragment was harvested from pHEN1-ScFv and inserted into pCANTAB5E, and the recombinant plasmid DNA was used to transform competent host XL1-Blue. After induction with IPTG for 20 hrs, the expressed HBsAg-ScFv in the supernantant of XL1-Blue was precipitated with 50% ammonium sulfated and demonstrated the molecular weight is about 28kDa by SDS-PAGE. The reactivity and specificity have been confirmed by enzyme-linked immuno-sorbent assay (ELISA). The identification and expression of HBsAg-ScFv from E.coli were successfully achieved. |
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