胆盐水解酶基因在毕赤酵母中的表达  

Expression of Bile Salt Hydrolase Gene in Pichia pastoris

作　　者：于长青[1] 战媛媛[1] 王长远[1] 

机构地区：[1]黑龙江八一农垦大学食品学院,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2010年第9期949-952,共4页Chinese Journal of Biologicals

基　　金：大庆市科技局攻关项目(SGG2007-031);大庆市高新区创新基金资助项目(DQGX08YF036);校博士启动基金项目资助(校启D2009-4)

摘　　要：目的在毕赤酵母中表达胆盐水解酶(Bile salt hydrolase,BSH)基因。方法以重组质粒pMD18-BSH为模板,PCR扩增BSH基因,克隆至毕赤酵母表达载体pGAPZαA上,经AvrⅡ线性化,电转化至毕赤酵母SMD1168,PCR筛选阳性重组子,诱导表达,表达产物经Western blot进行分析。结果重组表达质粒pGAPZαA-BSH经双酶切及测序证明构建正确;经PCR鉴定阳性的重组酵母菌的诱导表达产物经SDS-PAGE分析,可见相对分子质量约35000的目的蛋白表达条带;表达的重组蛋白可与兔抗植物乳杆菌多克隆抗体发生特异性反应。结论在毕赤酵母中成功表达了BSH,为BSH结构与相关功能及高效降解胆固醇的基因工程菌株的研究奠定了基础。Objective To express bile salt hydrolase（BSH）gene in Pichia pastoris.Methods BSH gene was amplified by PCR using recombinant plasmid pMD18-BSH as a template,and cloned into expression vector pGAPZαA.The constructed recombinant plasmid pGAPZαA-BSH was linearized with AvrⅡ,then transformed to P.pastoris SMD1168 by electrotransformation.Positive recombinants were screened by PCR for expression under induction.The expressed product was analyzed by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pGAPZαA-BSH was constructed correctly.SDS-PAGE showed protein band with a relative molecular mass of about 35 000.The expressed protein showed specific reaction with rabbit polyclonal antibody against Lactobacillus plantarum.Conclusion BSH was successfully expressed in P.pastoris,which laid a foundation of further study on the structure and function of BSH as well as recombinant bacterial strain highly degrading cholesterol.

关 键 词：胆盐水解酶 毕赤酵母 基因表达 

分 类 号：Q556[生物学—生物化学] Q786