【摘要】 目的:用酵母表达重组人平滑肌22α(SM22α)蛋白,制备兔抗人SM22α多克隆抗体。方法:利用PCR从pGEM3z-SM22α质粒扩增得到人SM22α基因编码区,重组至pPIC9构建酵母表达载体,转染巴斯德毕赤酵母,进行分泌型表达。表达产物经分步盐析和CM-纤维素柱层析纯化后,免疫家兔,制备兔抗人SM22α多克隆抗体。结果:所构建的pPIC9-SM22α酵母表达载体转染酵母感受态细胞后,外源性SM22α可整合至酵母染色体中,经甲醇诱导84 h,可实现SM22α的高效表达与分泌。用70%硫酸铵处理上清液,收集沉淀进行离子交换层析纯化后,经变性的聚丙烯酰胺凝胶电泳(SDS-PAGE)可见一分子量为22 kD的单一区带。用该纯化产物免疫家兔制备的兔抗人SM22α抗血清可用于检测人或大鼠血管壁中SM22α的表达水平。结论:重组人SM22α可在巴斯德毕赤酵母中进行高效表达与分泌,纯化蛋白可用于抗体制备,为研究SM22α功能提供了检测工具。更多还原

【Abstract】 Aim: The recombinant human smooth muscle 22 alpha(SM22α)was expressed by using Pichia pastoris.Methods: Using pGEM3z-SM22α as the template,SM22α coding region was amplified by PCR,and was inserted the expression vector pPIC9.Then the recombinant plasmid pPIC9-SM22α was transfected into Pichia pastoris.The products induced by methanol were precipitated by ammonium sulfate,then CM-cellulose chromatography was performed for SM22α.Polyclonal antibody against SM22α was produced by immunizing a rabbit with purified recombinant SM22α.Results: The positive clone with SM22α got high output at 84 hours after induction by methanol.The SM22α prepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE.Polyclonal antibody against SM22α could detect the SM22α expression in human or rat vascular walls.Conclusion: High-level expression of SM22α is successfully achieved in Pichia pastoris.Antibody against SM22α can be used to explore the function of SM22α.更多还原