鸡柔嫩艾美耳球虫HN株3-1E基因的原核表达与鉴定

Expression and identification of 3-1E of Eimeria tenella HN strain

根据已报道的柔嫩艾美耳球虫3-1E基因序列设计引物,以孢子化卵囊总RNA为模板,用RT-PCR方法扩增得到1条特异片段,将扩增产物克隆至p MD-18T载体,转化感受态菌JM109,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与已发表的国内外相关虫株的3-1E基因序列比较,核苷酸的同源性均在99.2%-99.8%,氨基酸的同源性均在98.2%-100%。然后将重组质粒和表达载体p GEX-4T-1分别用Xho I和EcoR I酶切后构建重组表达载体p GEX-3-1E,并将其转化入大肠杆菌BL21中,提取质粒经酶切和PCR鉴定正确后,用IPTG诱导表达。表达产物经SDS-PAGE和Western blot检测显示,3-1E基因在大肠杆菌中成功表达,融合蛋白的分子量为44.7 ku,诱导表达5 h的蛋白表达量可达到30%以上。

The 3-1E gene of Eimeria tenella HN strain was amplified by RT-PCR with a pair of primers designed according to the published sequences. Then the amplicon was cloned into pMD18-T easy vector, sequenced and analyzed. Compared with other related strains, the similarities of nucleotide sequences were from 99. 2% to 99. 8% , and the similarities of amino-acid sequences were from 98.2% to 100%. The gene fragment was ligated to the prokaryotic expression vector pGEX-4T-1 and expressed in Escherichia coli BL21 by IPTG induction. SDS-PAGE and Western-blotting analyses showed that the relative molecular weights of the fusion protein was measured approximately 44.7 ku, and the yield reached 30% of the total protein after induced by IPTG for 5 h.