人SM22α在巴斯德毕赤酵母中的表达、纯化及抗体制备

EXPRESSION,PURIFICATION AND ANTIBODY PREPARATION OF RECOMBINANT HUMAN SM22α

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。

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.