鸡柔嫩艾美耳球虫疫苗候选基因在大肠杆菌中的表达

目的　构建柔嫩艾美耳球虫子孢子表面抗原原核表达载体 ,并且在大肠杆菌中表达。　方法　将本室构建的pMD Mz5 7克隆质粒酶切回收目的片段定向亚克隆到pET 2 8b( )载体上 ,构建成原核表达载体pET 2 8b Mz5 7,酶切鉴定正确后 ,在EscherichiacoliBL2 1(DE3 )中用IPTG诱导表达 ,并经SDS PAGE及Westernblotting鉴定。　结果　成功构建了目的抗原基因的原核重组表达质粒pET 2 8b Mz5 7,IPTG诱导表达该融合蛋白 ,SDS PAGE电泳表明 ,其能表达一分子质量约为 3 7ku的融合蛋白 ,与预测分子质量相符 ,最佳反应条件为 1mol LIPTG诱导 4h后表达量最高。薄层扫描显示表达的融合蛋白占菌体蛋白总量的 18% ,Westernblotting结果显示 ,表达产物可被抗柔嫩艾美尔球虫的多克隆抗体识别 ,说明该融合蛋白具有很好的反应原性。　结论　在原核细胞融合表达Mz5 7成功 ,为鸡球虫病重组苗的研究奠定了基础。

EXPRESSION OF THE POTENTIAL VACCINE ANTIGEN OF EIMERIA TENELLA IN ESCHERICHIA COLI

Objective To construct the prokaryotic expression vector of the sporozoite surface antigen gene of Eimeria tenella GZ strain and expression in Escherichia coli. Methods The pMD-Mz5-7 plasmids were subcloned into the expression vector pET-28b( ) and constructed the recombinant plasmids pET-28b-Mz5-7 and then expressed in E. coli DE3 by inducing of IPTG. The fusion product was identified by SDS-PAGE gel electrophoresis and Western blotting were proceeded with anti-Mzp5-7 sera from chicken. Results The gene could synthesis a fusion protein at high levels. Peaks of target protein was achieved at 4 h after adding 1 mmol/L IPTG. Further more,the protein could react with the speciality anti-Mz5-7 sera. Conclusion The fusion protein of Mz5-7 was expressed successfully in prokaryotic cells and it laid a foundation of the recombinant subunit vaccine.