| 生精相关基因mTSARG3的原核表达和抗体制备 |
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| 引用本文： | 刘刚,卢光琇.生精相关基因mTSARG3的原核表达和抗体制备[J].南方医科大学学报,2010,30(9):2070. |
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| 作者姓名： | 刘刚 卢光琇 |
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| 作者单位： | 中南大学生殖与干细胞工程研究所,湖南,长沙,410078 |
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| 基金项目： | 教育部博士学科点新教师基金 |
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| 摘 要： | 目的 构建小鼠生精相关基因pGEX-KG/mTSARG3重组载体,进行原核表达和多克隆抗体制备.方法 应用RT-PCR从小鼠睾丸cDNA文库中扩增mTSARG3的开放阅读框,T-A克隆后将mTSARG3插入到原核表达载体pGEX-KG中,经测序鉴定后将重组质粒转入宿主菌E.coliBL21,IPTG诱导表达GST/mTSARG3融合蛋白,采用SDS-PAGE和Westernblotting对融合蛋白进行分析和鉴定.以融合蛋白为免疫原免疫家兔,制备抗mTSARG3多克隆抗体并进行Westernblotting鉴定.结果 成功构建了pGEX-KG/mTSARG3重组质粒,测序结果与预期一致;转化重组质粒的E-coliBL21经IPTG37℃诱导4h后高效表达GST/mTSARG3融合蛋白:Westernblotting实验结果显示制备抗体可与原核表达融合蛋白特异性结合.结论 成功进行了mTSARG3基因的原核表达和多克隆抗体制备,为下一步的功能研究奠定基础.
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| 关 键 词： | 基因克隆 蛋白表达 多克隆抗体 精子发生 |
Construction of pGEX-KG/mTSARG3 recombinant vector and preparation of anti-mTSARG3 polyclonal antibody |
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| LIU Gang,LU Guang-xiu.Construction of pGEX-KG/mTSARG3 recombinant vector and preparation of anti-mTSARG3 polyclonal antibody[J].Journal of Southern Medical University,2010,30(9):2070. |
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| Authors: | LIU Gang LU Guang-xiu |
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| Institution: | LIU Gang,LU Guang-xiu Institute of Reproduction and Stem Cell Engineering,Central South University,Changsha 410078,China |
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| Abstract: | Objective To construct pGEX-KG/mTSARG3 recombinant vectorand prepare the fusion protein to obtain rabbit polyclonal antibody against mTSARG3.Methods The open reading frame(ORF) of mTSARG3 gene was amplified from mouse testis cDNA library by PCR.The products were cloned into pGEM-T Easy vectors and sequenced.The recombinant plasmids were digested by EcoRI and SalI and subcloned into PGEX-KG vector.After identification by DNA sequence analysis,the recombinant plasmids were transformed into component E.coli BL... |
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| Keywords: | mTSARG3 |
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