猪血清白蛋白基因的克隆及其在毕赤酵母中的分泌表达

Cloning and Secretive Expression of Porcine Serum Albumin Gene in Pichia pastoris

目的克隆猪血清白蛋白(PSA)基因,并在毕赤酵母中分泌表达。方法Trizol法提取新鲜猪肝组织总RNA,经RT-PCR扩增PSA全长cDNA,克隆至酵母分泌型表达载体pPICZaC,构建重组表达质粒pPICZaC-PSA,电转化至毕赤酵母X33,甲醇诱导表达,表达产物进行SDS-PAGE和Western blot分析。结果重组表达质粒pPICZaC-PSA经双酶切及测序鉴定正确,表达的重组蛋白经SDS-PAGE分析,在相对分子质量约69500处可见特异条带,表达量为菌体总蛋白的29.5%,紫外吸收法测定蛋白含量为0.06mg/ml,并可与PSA多抗发生特异性反应。结论已成功克隆了PSA基因,并在毕赤酵母中获得表达。

Objective To clone porcine serum albumin （PSA）gene and express in a secretive form in Pichia pastoris. Methods Total RNA was extracted from fresh porcine liver by Trizol method and used as a template for amplification of full-length cDNA of PSA by RT-PCR. The amplified gene was cloned into secretive expression vector pPICZaC, and the constructed recombinant plasmid pPICZaC-PSA was transformed into P. pastoris X33 by electrotransformation for expression under induction of methanol. The expressed product was identified by SDS-PAGE and Western blot. Results Both restriction analysis and sequencing proved that re-combinant plasmid pPICZaC-PSA was constructed correctly. SDS-PAGE showed that the expressed protein, with a relative molecular mass of about 69 500, contained 29. 5% of total somatic protein. The protein content of expressed product was 0. 06 mg / ml as proved by ultraviolet absorption. Western blot showed specific reaction of expressed protein with polyclonal antibody against PSA...更多. Conclusion PSA gene was successfully cloned and expressed in P. pastoris.