| 小鼠B7-H4原核表达载体的构建、表达及多克隆抗体的制备 |
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| 引用本文： | 肖德乾,肖欢,胡国艳,那志萍,郑淑华,刘伟,张良清,徐军发.小鼠B7-H4原核表达载体的构建、表达及多克隆抗体的制备[J].湛江医学院学报,2009,27(5):481-484. |
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| 作者姓名： | 肖德乾 肖欢 胡国艳 那志萍 郑淑华 刘伟 张良清 徐军发 |
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| 作者单位： | [1]广东医学院检验医学研究所,广东东莞523808 [2]广东医学院附属医院麻醉科,广东湛江524001 |
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| 基金项目： | 广东省科学事业费计划项目，广东医学院博士基金项目，广东省医学科研课题 |
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| 摘 要： | 目的探讨小鼠B7-H4原核表达载体的构建、表达及多克隆抗体的制备。方法采用特异性引物扩增小鼠B7-H4（mB7-H4）胞外功能区DNA,经酶切、拼接构建原核表达载体pET28a-mB7-H4。将构建的重组表达质粒转化入E.coliBL21（DE3）菌株,采用IPTG诱导表达、Ni-NTA柱亲和层析纯化目的蛋白、SDS-PAGE分析蛋白纯度。将纯化的目的蛋白免疫家兔制备多克隆抗体,并对其进行纯化及鉴定。结果序列测定证实了构建的pET28a-mB7-H4重组表达载体含有mB7-H4编码序列,其序列分析与GenBank中公布序列对比一致,质粒在E.coli中诱导表达相对分子质量（Mr）为26.5 KD的目的蛋白,SDS-PAGE分析表明纯化后的目的蛋白达到电泳纯。双向琼脂扩散法检测抗体效价为1∶16,ELISA法检测抗体效价为1∶12 800,Western blot分析显示抗体能特异性结合mB7-H4。结论成功制备了高表达重组mB7-H4蛋白的原核表达载体及高效价抗mB7-H4多克隆抗体,为进一步研究B7-H4的功能奠定了基础。
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| 关 键 词： | B7-H4 原核表达载体 基因表达 多克隆抗体 |
Construction and expression of a prokaryotic expression vector for murine B7-H4 and preparation of a polyclonal antibody |
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| XIAO De-qian,XIAO Huan,HU Guo-yan,NA Zhi-ping,ZHENG Shu-hua,LIU Wei,ZHANG Liang-qing,XU Jun-fa.Construction and expression of a prokaryotic expression vector for murine B7-H4 and preparation of a polyclonal antibody[J].Journal of Guangdong Medical College,2009,27(5):481-484. |
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| Authors: | XIAO De-qian XIAO Huan HU Guo-yan NA Zhi-ping ZHENG Shu-hua LIU Wei ZHANG Liang-qing XU Jun-fa |
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| Institution: | XIAO De-qian,XIAO Huan,HU Guo-yan,NA Zhi-ping,ZHENG Shu-hua,LIU Wei,ZHANG Liang-qing,XU Jun-fa(Institute of Clinical Laboratory Medicine,Guangdong Medical College,Dongguan 523808,China) |
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| Abstract: | Objective To study the construction and expression of a prokaryotic expression vector for murine B7-H4 and preparation of a polyclonal antibody.Methods Murine B7-H4(mB7-H4)gene was amplified with specific primers.The PCR product was digested with enzymes and then ligated to reconstruct recombinant prokaryotic expression vector pET28a-mB7-H4 that expresses the mouse B7-H4 and 6 His fusion protein.The expression vector was transformed into E.coli BL21(DE3) and induced with IPTG to express mB7-H4 fusion protei... |
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| Keywords: | B7-H4 |
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