微小隐孢子虫黏附相关蛋白基因CP21的原核表达及鉴定

摘要

The research was conducted to clone the related genes of adhesion-related protein CP21 of Cryptosporidium parvum, construct its recombinant plasmid and identify the fusion protein. The DNA containing CP21 gene was extracted from phages and the CP21 gene was amplified by PCR. Then prokaryotic expression plasmids pET-28a-CP21 was constructed. The expression of CP21 fusion protein was induced by IPTG in E . Coli BL2KDE3) system and then identified by SDS-PAGE. The mice were immunized with the CP21 fusion protein for preparing specific poly-clonal antibody against the protein, and then the CP21 protein was localized in C. parvum. The protein of CP21 was expressed as fusion protein, 25 kDa. Western blotting results indicated that CP21 fusion protein could specially reacted with polyclonal antibody against the CP21 of C. parvum which expressed in sporozoite and oocyst stage. The expression products localized on the surface membrane of sporozoite and oocyst wall. CP21 was a adhesion proteins related to invasion mechanisms which could be used as a effective target site to prevent and cure cryptosporidiosis.%本研究旨在克隆微小隐孢子虫黏附相关蛋白基因CP21开放阅读框,构建其原核表达质粒,并对获得的融合蛋白进行鉴定.从含CP21基因的噬菌体中提取模板DNA,PCR扩增基因片段,构建pET-28a-CP21原核表达质粒,转化至埃希氏大肠杆菌BL21 (DE3)中,IPTG诱导表达,表达产物经SDS-PAGE和Western blotting分析鉴定.用原核表达蛋白免疫小鼠,制备抗重组蛋白多抗,对蛋白进行定位.结果表明成功扩增出CP21基因,并构建原核表达质粒,转化至大肠杆菌中表达出相对分子质量为25 ku的融合蛋白,Western blotting证明表达的CP21融合蛋白能与抗C.parvum多克隆抗体产生特异性反应.免疫荧光结果表明:CP21基因在子孢子和卵囊期均表达,且表达产物位于子孢子表膜和卵囊壁表面.CP21是一种与侵入机制有关的黏附相关蛋白.研究结果为微小隐孢子虫的免疫学诊断及疫苗研制奠定了基础.