毒害艾美耳球虫(Eimeria necatrix)广东株微线蛋白-2基因在大肠杆菌的表达

根据克隆的毒害艾美耳球虫(Eimeria necatrix)广东株微线蛋白-2基因(EnMIC-2(Gd))的cDNA序列设计特异性引物，用PCR方法扩增其阅读框架(ORF)后，克隆至质粒表达载体pET-32a( )，成功构建了重组表达质粒pET-32a( )-EnMIC-2。用CaCl2法将其转化至宿主细菌E.cliBL21(DE3)，并用IPTG成功诱导了EnMIC-2重组抗原在大肠杆菌表达。表达的重组蛋白约占菌体总蛋白的10．8％，其相对分子质量约为55000。重组蛋白经SDS-AGE分析后，用E．necatrix(广东株)感染鸡的高免血清进行Western Blotting分析，结果为阳性。

Expressing Microneme-2 Gene of E. necatrix in E. coli

The ORF of Microneme-2 gene from E.necatrix Guangdong strain was amplified by PCR using the specific pri-mers designed according to the sequences of EnMIC-2(Gd) gene cloned in our laboratory,and its ligated products with vector pET-32a(+) were transformed to E.coli BL21(DE3) by CaCl_2 method.The transformants were identfied by PCR amplification and endonuclease digestion.The sequences of positive clone were analyzed,and the recombinant protein was induced to be expressed by 1 mmol/L IPTG in vitro.The molecular weight of EnMIC-2 recombinant protein is about (55 000),the amount of recombinant protein in total bacteria protein to be some 10.8%.Western blotting result of purified EnMIC-2 recombinant protein was positive when chicken hyperimmune serum of E.necatrix was used as probe.