牛微小隐孢子虫表面抗原CP15基因原核表达及其表达产物的鉴定

Prokaryotic Expression and Identification of Cryptosporidium parvum Surface Antigen CP15 Gene in Escherichia coli BL21

试验旨在构建微小隐孢子虫表面抗原CP15基因原核表达载体并获得重组CP15蛋白。试验以已知重组质粒pMD-CP15为模板,PCR方法扩增出CP15基因片段,并亚克隆到pGEX4T-3,构建了在E.coli BL21中GST融合表达载体pGEX-CP15;经1mmol/L IPTG进行诱导表达获得目的蛋白,其大小采用SDS-PAGE电泳和Western blotting进行鉴定。结果表明,扩增出约390bp的微小隐孢子虫表面抗原CP15基因片段并成功构建pGEX-CP15质粒,表达出分子质量为42ku的融合蛋白,与推导的理论值相符。Western blotting显示该蛋白能被GST血清识别。牛微小隐孢子虫表面抗原CP15基因在大肠杆菌中得到了高效表达。

To construct a recombinant plasmid contain CP15 gene of Cryptosporidium parvum （C. parvum） and obtain re- combinant protein. The surface antigen CP15 gene fragment of C. parvum was amplified from plasmid pMD-CP15 by PCR,and subcloned into pGEX 4T-3. The fusion express recombinant vector pGEX-CP15 was constructed in E. coli BL21. The recombi- nant protein was induced by 1 mmol/L IPTG and identified by SDS-PAGE and Western blotting. The CP15 gene fragment was amplified correctly as the size of gene was about 390 bp, and the recombinant plasmid pGEX-CP15 was constructed. The pro- tein band with a molecular weight of 42 ku was detected on SDS-PAGE, which totally same with the theoretical size. The ex- pressed protein was identified by Western blotting performed with GST serum. The fusion protein of CP15 was highly ex- pressed in E. coli.