柔嫩艾美耳球虫表面抗原(EtSAG)基因的分离及鉴定

ISOLATION AND IDENTIFICATION OF A NOVEL SURFACE ANTIGEN GENE OF EIMERIA TENELLA

利用本实验室前期获得的柔嫩艾美耳球虫(Eimeria tenella)孢子化卵囊和未孢子化卵囊差异表达ESTs序列,选取编号为BW4-C03的孢子化卵囊,采用RACE技术,获得该基因全长序列。经BLAST分析,该序列与柔嫩艾美耳球虫表面抗原有72%以上的同源性,命名为EtSAG。利用荧光定量PCR(Real-time PCR)检测发现该基因在孢子化卵囊的转录拷贝数最高,且随着孢子化时间的延长,转录拷贝数逐渐增加。采用原核表达载体pET-28C表达该基因,得到的融合蛋白大小约为36 kDa,符合预期大小。经Western blot分析,该重组蛋白可被兔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。

A novel surface antigen gene of Eimeria tenella,designated as BW4-C03,was cloned by RACE technology based on ESTs of differentially expressed genes of unsporulated oocysts and sporulated oocysts.Blast searches found that BW4-C03 sequence shared more than 72% homology with that of the previously publishd surface antigen of E.tenella.Real-time PCR revealed that BW4-C03 was expressed at the higher level in sporulated oocysts than other developmental stages（unsporulated oocysts,sporozoites and merozoites）.Then,BW4-C03 was cloned to the prokaryotic expression vector pET-28c（＋）.The recombinant protein of interest was expressed with induction by IPTG as visualized in SDS-PAGE.The fusion protein was specifically recognized by polyclonal antibodies against E.tenella in Western blotting.These results provided useful information for future study on the biological activities of the novel surface antigen.