巨型住肉孢子虫烯醇酶基因克隆与表达

Clone and Expression of Enolase in Sarcocystis gigantea

巨型住肉孢子虫(Sarcocystis gigantea)是羊体内一种常见寄生虫,多寄生于羊横纹肌内形成包囊,导致羊肉大量废弃,给畜牧业带来巨大经济损失。本研究尝试筛选能有效诊断住肉孢子虫感染的抗原。通过免疫印迹及质谱分析筛选到巨型住肉孢子虫候选诊断抗原烯醇酶,利用染色体步移技术扩增两侧翼未知序列并表达重组蛋白。应用生物信息学分析和免疫印迹对该蛋白诊断价值进行评价。结果筛选到的候选蛋白为巨型住肉孢子虫烯醇酶(SgENO),克隆得到1 181 bp的基因并获得重组蛋白rSgENO。对rSgENO应用进行初步评价,发现其与其他顶复亚门原虫的烯醇酶氨基酸相似性均较高(71%～92.1%),且能被弓形虫和新孢子虫阳性血清所识别,具有交叉反应性。本研究克隆并表达了巨型住肉孢子虫烯醇酶,后期评价发现该重组蛋白与新孢子虫和弓形虫有较强的交叉反应性,不能用于羊住肉孢子虫的血清学诊断,但有成为疫苗候选分子的潜力。

Sarcocystis gigantea is a globally ubiquitous pathogen that infects sheep. The formation of cyst in striated muscle induces a largely waste of commercial mutton which causes livestock husbandry losses. In this study, we tried to explore the special antigens for serological diagnosis of sarcocystosis. We screened a series of candidate antigens in S. gigantea through Western blot and mass spectrum identification. Whereafter, Genome Walking technology was applied to acquire two flanking sequences of these antigens. Ultimately, purify recombinant proteins was used to evaluate diagnostic value by bioinformatics analysis and Western blot. Results were as follows: Enolase of S. gigantea was finally supposed to a candidate diagnostic antigen, named SgENO. Subsequently, 1 181 bp expressive sequence of SgENO was cloned, and recombinant protein rSgENO was obtained. Nevertheless, it was found to have high homology with enolase amino acids of other Apicomplexa (71%-92.1%), rSgENO could cross react with Toxoplasma gondii and Neospora caninum positive serum. We cloned and expressed the S. gigantea enolase, and the recombinant protein had a cross reaction with other parasitic protozoa positive serum, which made it an irrelevant antigen for Sarcocystis specific diagnose. However, this protein may be a candidate antigen for vaccines.