泰泽隐孢子虫CP15基因的原核表达及抗血清制备

摘要

将泰泽隐孢子虫(Cryptosporidium tyzzeri)CP15基因从pMD18-T-CP15重组质粒中切下,连接至pET-28a(+)原核表达载体,经酶切和测序鉴定正确后,转化到大肠埃希菌BL21(DE3)中进行诱导表达,表达产物进行SDS-PAGE和Western blot分析,并将纯化的重组蛋白免疫ICR小鼠制备多克隆抗体.结果显示,重组表达载体pET-28a-CP15在大肠杆菌中以可溶形式表达,表达蛋白分子量约为17 kDa.Western blot显示重组蛋白能被泰泽隐孢子虫感染小鼠血清所识别.ELISA结果显示重组蛋白免疫小鼠血清能和泰泽隐孢子虫卵囊可溶性抗原特异性反应,表明获得的重组蛋白具有较好的抗原性.%In order to express CP15 gene of Cryptosporidium tyzzeri in prokaryotic expression system, CP15 gene was digested from recombinant pMD18-T-CP15 and cloned to the prokaryotic expression vector pET-28a(+). The recombinant pET-28a-CP15 was identified with restrict enzyme digestion and sequencing and then transfected into E. coli BL21 (DE3). The protein expression was induced with IPTG and visualized in SDS-PAGE and Western blot. The results showed that the plasmid pET-28a-CP15 was expressed in E. coli BL21 (DE3) and recombinant CP15 protein (rCP15) was soluble. The recombinant rCP15 was measured to be about 17 kDa in SDS-PAGE and reacted with positive mouse antiserum in Western blot. The purified rCP15 was used to immunize ICR mice to prepare polyclonal antibodies, which reacted in ELISA with soluble antigen of C. tyzzeri oocysts. These results showed that the rCP15 had a good antigenicity.