皮蝇素C蛋白在毕赤酵母中的表达及纯化

Expression and Purification of Recombinant Hypodermin C in Pichia pastoris

摘要:

利用PCR技术从重组质粒pGEM－T/HC中扩增HC cDNA片段，克隆到pPIC9k毕赤酵母表达载体中，获得重组质粒pPIC9k-HC，重组质粒线性化后电激转化入毕赤酵母GS115菌株中，重组子经G418筛选、PCR鉴定得到含外源基因的重组子，然后在含0.5%甲醇的培养基中诱导产生目的蛋白，经SDS－PAGE检测发现表达蛋白质的相对分子量约为28kD，表达量约为121 mg/L，表达上清经超滤浓缩和阴离子交换层析初步纯化，所得纯化产物经Western-blot表明具有与兔抗HC血清特异性结合的能力；明胶电泳检测显示具有水解酶活性，为今后进行大规模的牛皮蝇蛆病的调查提供了一种生产大量廉价抗原的方法。

Abstract:

Hypodermin C( HC) cDNA was amplified from recombinant pGEM－T/HC， cloned in frame with the signal sequence in yeast vector pPIC9k. The plasmid was linerarized and transformed into Pichia pastoris GS115 strain by electroporation method. Recombinant strain was screened by G418 resistant, and further confirmed by PCR. The recombinant strain which contains insert was induced in the medium containing 0.5% methanol. The supernatant was collected and then purified by anion exchange chromatography. SDS-PAGE indicated that the target protein is around 28kD. Western-blot showed it can react with rabbit-anti HC serum. Gelatin substrate SDS-PAGE displayed it had enzyme activity. Provided a method to produce enough antigens for carrying out extensive immunological analyses.