柔嫩艾美耳球虫烯醇化酶基因的克隆表达和抗原性分析

Cloning, expression and antigenic analysis of enolase gene of Eimeria tenella

从纯化的柔嫩艾美耳球虫第二代裂殖子中提取总RNA,应用RT-PCR技术克隆获得了烯醇化酶基因(Gen Bank Accession No.GU169333)。烯醇化酶基因开放阅读框长1 338 bp,编码445个氨基酸。应用DNAstar软件对氨基酸序列的亲水区域和疏水区域的分布、抗原指数及T细胞基序进行分析,结果显示该蛋白质亲水性较强,抗原指数高,含较多T细胞表位。同时构建了烯醇化酶原核表达重组质粒p ET-28a(+)-ENO。用IPTG对重组质粒p ET-28a(+)-ENO进行诱导表达,SDS-PAGE结果显示表达的融合蛋白大小约为5.17×104,主要存在于裂解上清中。Western blotting结果显示该重组蛋白可被抗柔嫩艾美耳球虫的多克隆抗体识别,表明该蛋白质具有较好的免疫原性。

Enolase gene of Eimeria tenella （ GenBank Accession No. GU169333） （ EtENO） was amplified from total RNA extracted from E. tenella second-generation merozoites by RT-PCR. The sequence of EtENO contains an open reading frame of 1338 bp and encodes 445 amino acid. DNAstar software analysis showed that EtENO has high antigenic index, lots of hydrophilicity plots and T cell motif. The recombinant plasmid pET-28a （ ＋ ）-ENO was constructed and transformed into Escherichia coli BL21（DE3） for expres-sion. SDS-PAGE indicated that the fusion protein （5.17＆#215; 104 ） was expressed in the supernatant after induction by IPTG. Western blotting revealed that the protein was spe-cifically recognized by polyclonal antibodies against E. tenella, suggesting that the fusion protein is antigenic.