中华泰勒虫MPSP基因的原核表达及反应原性分析

Prokaryotic expression of Theileria sinensis MPSP gene and reactionogenicity analysis of the expressed protein

用生物信息学方法,对中华泰勒虫的梨浆虫主要表面蛋白(major piroplasm surface proteins,MPSP)基因的核苷酸序列及编码蛋白的氨基酸序列进行了分析,并对该蛋白的抗原表位、信号肽以及跨膜区进行了预测。设计特异性引物,对中华泰勒虫MPSP基因进行了克隆和原核表达,并对融合蛋白的反应原性进行了分析。SDS-PAGE结果显示,融合蛋白的分子质量约为38ku,分布于上清中。Western-blot试验表明,融合蛋白仅与抗His标签蛋白的单克隆抗体、中华泰勒虫阳性血清、瑟氏泰勒虫阳性血清、环形泰勒虫阳性血清发生反应,而与其他泰勒虫、巴贝斯虫和绵羊无形体阳性血清不发生反应。这些结果表明,中华泰勒虫MPSP可作为ELISA诊断方法的候选抗原。

Nucleotide and putative amino acid sequences of the gene coding major piroplasm surface proteins(MPSP)of Theileria sinensis were analyzed,and then the proteins epitope,signal peptide and transmembrane domain were predicted using bioinformatics methods.Specific primers were designed and MPSP gene was expressed in prokaryotic expression system followed by analyzing the reactionogenicity of fusion protein by Western-blot.The SDS-PAGE result showed that the fusion protein in the supernatant was approximately 38ku in size,and the Western-blot result showed that the fusion protein had strong cross-reactions with anti-His tag protein and the positive sera from T.sinensis,Theileria sergenti and Theileria annulata,but no cross-reaction with positive sera against other Babesia,Theileriaand Anaplasma ovis species,respectively.It suggests that T.sinensis MPSP may be used as a diagnostic antigen to develop ELISA.