微小隐孢子虫CP15抗原基因原核表达载体的构建及在大肠杆菌中的表达

EXPRESSION OF CP15 FUSION PROTEIN ON SPOROZOITES OF CRYPTOSPORIDIUM PARVUM IN ESCHERICHIA COLI

目的 构建微小隐孢子虫子孢子表面抗原CP15的原核表达载体 ,并且在大肠杆菌中表达。 方法 用HindⅢ和EcoRⅠ酶分别从pET 2 8a(+ )和 pMD18 T 15质粒中酶切得到线性片段和CP15基因片段 ,然后用T4DNA连接酶连接 ,构建重组的CP15的原核表达载体 ,在大肠杆菌BL2 1(DE3 )中表达 ,并用SDS PAGE、ELISA和Westernblotting进行鉴定。 结果 构建了CP15的原核表达载体 ,得到了一分子质量约为 15ku的融合蛋白 ,占大肠杆菌总蛋白的 3 8%。 结论 CP15融合蛋白在大肠杆菌中得到了高效表达。

Objective To construct expression vector of CP15 on sporozoites of Cryptosporidium parvum and express it in E. coli . Methods A linear fragment of pET-28a(+) and CP15 gene of C. parvum were obtained from the plasmids of pET-28a(+) and pMD18-T-15 with Hind Ⅲ and EcoRⅠdisgested. The expression vector of CP15 was constructed by T_ 4 ligase and transformed into BL21(DE3) strain cells of E. coli for expression. The expression products were analyzed by SDS-PAGE, ELISA and Western blotting. Results The expression vector of CP15 was constructed. Distinct protein band with a molecular weight of 15 ku was detected on SDS-PAGE and its antigenicity was confirmed by ELISA and Western blotting. Conclusion Efficient expression of CP15 fusion protein has been achieved in E.coli .