论文部分内容阅读
为了研究钙蛋白酶系统在细胞发育及其它生理过程的功能 .应用 PCR从鼠钙蛋白酶抑制蛋白 ( calpastatin) c DNA中扩增了保守的具有功能的结构域 ( 40 4 bp) ,克隆于 p GEX- KG载体 .重组质粒 p GEX- Calp4在大肠杆菌中经 IPTG诱导可表达分子量约 4 5 k D融合蛋白 GST- Galp4 .诱导表达后的菌体超声裂解液经谷胱甘肽 - Sepharose4 B亲和层析柱得到纯化的 GST- Calp4融合蛋白 ,纯度达电泳纯 .纯化的 GST- Calp4免疫兔 8周后 ,抗血清的效价达 1∶ 64 .Western- blot分析表明制备的抗血清确实可以与肌细胞中分子量为 1 4 0 k D左右蛋白 (亦即完整 calpastatin)发生特异的免疫交叉反应 ,此表明实验获得了高特异性多克隆抗体
In order to study the function of calpain system in cell development and other physiological processes, a conserved functional domain (40 4 bp) was amplified from calpastatin c DNA by PCR and cloned into pGEX- KG vector.The recombinant plasmid pGEX-Calp4 was induced by IPTG in Escherichia coli to express the fusion protein GST-Galp4 with a molecular weight of about 4 5 kD.The bacterial lysate after induced by glutathione-Sepharose 4B affinity chromatography Purified GST-Calp4 fusion protein was purified by electrophoresis on the analytical column, and the purity of the purified GST-Calp4 immunized rabbit was 8 weeks after antiserum titer was 1:64 .Western-blot analysis showed that the prepared antiserum could indeed bind with muscle Specific immune cross-reactivity of cells with a molecular weight of about 140 kD (ie, intact calpastatin) demonstrated that the experiment resulted in the production of highly specific polyclonal antibodies