| 抗HIV-1 gp120单克隆抗体的制备及鉴定 |
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| 引用本文： | 张永臣,文剑,张亮,赵磊.抗HIV-1 gp120单克隆抗体的制备及鉴定[J].中国生化药物杂志,2010,31(1). |
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| 作者姓名： | 张永臣 文剑 张亮 赵磊 |
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| 作者单位： | 东南大学,附属第二医院,检验科,江苏,南京,210003 |
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| 基金项目： | 江苏省自然科学基金，南京市社会发展项目 |
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| 摘 要： | 目的制备抗HIV-1 gp120单克隆抗体,鉴定其特性,为进一步研制HIV治疗性抗体提供贮备。方法将HIV-1 gp120基因片段连接到经酶切的原核表达载体PEGX-4T-2中,利用大肠杆菌XL1-blue表达GST-HIV蛋白。表达的蛋白纯化后免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性克隆。重组免疫印迹分析(RIBA)和Western blot检测抗体特异性。结果构建的重组质粒的外源基因片段经测序与预期HIV-1 gp120抗原基因片段大小一致。纯化蛋白SDS-PAGE可见1条相对分子质量(Mr)32 000的表达蛋白条带。获得6株稳定分泌单克隆抗体的杂交瘤细胞株,其细胞培养上清效价为10-3,小鼠腹水效价为10-5,抗体亚型均为IgG1。RIBA结果显示6株单抗皆只与HIV-1 gp120抗原反应。Western blot结果显示在Mr32 000处可见1条单一目的条带。结论获得6株稳定分泌抗HIV-1 gp120单克隆抗体的杂交瘤细胞株,为进一步研制抗HIV治疗性抗体奠定了基础。
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| 关 键 词： | HIV-1gp120抗原 原核表达 单克隆抗体 杂交瘤细胞 |
Preparation and identification of anti-HIV-1 gp120 monoclonal antibody |
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| ZHANG Yong-chen,WEN Jian,ZHANG Liang,ZHAO Lei.Preparation and identification of anti-HIV-1 gp120 monoclonal antibody[J].Chinese Journal of Biochemical Pharmaceutics,2010,31(1). |
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| Authors: | ZHANG Yong-chen WEN Jian ZHANG Liang ZHAO Lei |
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| Institution: | ZHANG Yong-chen,WEN Jian,ZHANG Liang,ZHAO Lei(The N0.2 Hospital Affiliated to Southeast University,Nanjing 210003,China) |
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| Abstract: | Purpose To Prepare anti-HIV-1 gp120 monoclonal antibodies and to identify the specificity of antibodies in order to provide technique for preparing HIV remedial antibodies.Methods The gene fragment of HIV-1 gp120 was connected to PEGX-4T-2 prokaryotic expressing vector.The vector was cut by enzyme.GST-HIV protein was expressed by E.coli XL1-blue.The BALB/c mice were immunized with purified GST-HIV antigen,and then the fusion of mice spleen cell and myeloma cell SP2/0 was executed as the routine cell-fusion ... |
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| Keywords: | HIV-1 gp120 antigen prokaryotic expression monoclonal antibody hybridoma cell |
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