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

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

根据克隆的毒害艾美耳球虫 (Eimeria necatrix)广东株微线蛋白 - 2基因 (En MIC- 2 (Gd) )的 c DNA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 p ET- 32 a(+) ,成功构建了重组表达质粒 p ET-32 a(+) - En MIC- 2。用 Ca Cl2 法将其转化至宿主细菌 E.coli BL2 1(DE3) ,并用 IPTG成功诱导了 En MIC- 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 5 5 0 0 0。重组蛋白经 SDS- PAGE分析后 ,用 E.necatrix(广东株 )感染鸡的高免血清进行 Western Blotting分析 。

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.