钙蛋白酶抑制蛋白功能结构域Ⅳ在大肠杆菌中的表达、纯化及其抗血清的制备

Expression of Functional Domain Ⅳ of Calpastatin in E.coli and Preparation of Antiserum Against Purified GST-Calpastatin

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 。

Lots of researches have suggested that calpain system plays an important role in numerous cell functions,but its mechanism is still not clear now. In order to further research,the conservative domain Ⅳ,with inhibitory activity,was amplified by PCR and cloned into expression vector pGEX KG and transformed E.coli . After 3 h induced by IPTG,the expression of recombinant 45 kD GST Calp4 fusion protein was confirmed by SDS PAGE.GST Calp4 was purified from lysates with glutathione Sepharose 4B column.The purified GST Calp4 was mixed with Freund's complete or incomplete adjuvant,and then used to immunize rabbits.Eight weeks later,the fiter of antiserum was 1∶64.The IgG form was then purified by fractionation with (NH 4) 2SO 4.When extracted protein from muscle cells was applied for Western blot analysis,there is only one band around 140 kD was detected,which is the same as the molecular weight of complete calpastatin.