柔嫩艾美耳球虫HSP基因的克隆、表达及鉴定

Cloning, expression and analysis of HSP gene from Eimeria tenella

摘要:

为研究柔嫩艾美耳球虫热激蛋白(Heat shock proteins, HSPs)的生物学特性, 应用RACE和RT-PCR技术, 从柔嫩艾美耳球虫子孢子中首次克隆获得了EtHSP的全长cDNA(GenBank Accession No. FJ911605)。EtHSP包含一个1455 bp的开放阅读框, 编码484个氨基酸, 预测表达蛋白的分子量大小为53.5 kD。应用Real-time PCR对柔嫩艾美耳球虫不同发育阶段(未孢子化卵囊、孢子化卵囊、子孢子和裂殖子)表达量进行分析, 发现该基因在子孢子阶段的表达明显高于其他阶段。同时, 构建了原核表达重组质粒pET28a(+)-EtHSP, 转化到大肠杆菌BL21(DE3)中, 经IPTG诱导表达后, 对表达产物进行SDS-PAGE及Western blotting分析。结果显示, 重组质粒pET28a(+)-EtHSP在大肠杆菌中以包涵体形式表达, 经1 mmol/ L IPTG诱导6 h后的表达量最高, 该蛋白可被抗柔嫩艾美耳球虫的多克隆抗血清识别, 表明该蛋白具有较好的反应原性。本研究结果为进一步研究该基因的生物学功能奠定了基础。

Abstract:

In order to study the functions of the HSPs (Heat shock proteins) of Eimeria tenella, we cloned a novel gene (which designated EtHSP) coding HSP of Eimeria tenella by RT-PCR and RACE (Rapid-amplification of cDNA ends). The full-length cDNA sequence of EtHSP was 1802 bp, containing a 1455 bp ORF (Open reading frame) (GenBank Accession No. FJ911605) encoding a deduced protein of 484 amino acids. Real-time PCR revealed that the mRNA level of EtHSP was much higher in sporozoites of E. tenella than other developmental stages (unsporulated oocysts, sporulated oocysts and merozoites). We constructed the recombinant plasmids pET28a(+)-EtHSP , then transformed it into E. coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed in included bodies, with peak expression 6 h after induction by IPTG. Western blotting revealed that the protein was specifically recognized by polyclonal antibodies against E. tenella, showing that the fusion protein was native antigen.