马泰勒虫EMA1基因的克隆与生物学特性分析

Cloning and phylogenetic analysis of EMA1 gene of Theileria equi

根据GenBank上登录的马泰勒虫美国株(AB043618)裂殖子表面蛋白抗原1(EMA1)基因,设计了2对引物,采用PCR技术首次扩增出我国马泰勒虫肇源株EMA1基因。将该基因克隆到pMD19-T载体后,进行序列测定及分析。将鉴定为阳性的克隆重组到原核表达载体pET-30a后进行表达,并纯化表达的重组蛋白,进行Western-blotting试验,以鉴定其生物学活性。结果,EMA1基因长819bp,编码272个氨基酸,将该基因编码蛋白的氨基酸序列与GenBank中登录的11种已知梨形虫的相应氨基酸序列进行比较分析,马泰勒虫肇源分离株与已报道的马泰勒虫(马巴贝斯虫)亲缘关系最近,其次是环形泰勒虫和斑羚泰勒虫,而与东方泰勒虫、瑟氏泰勒虫的亲缘关系较远。表达的重组蛋白是分子质量约为38ku的融合蛋白,且表达产物能被马泰勒虫标准阳性血清识别。结果表明,马泰勒虫EMA1作为一种良好的抗原分子,在虫株的分类学研究、流行病学调查以及候选疫苗的研制中有着重要的意义。

To clarify the genetic background and molecular epidemiology of Theileria equi（T.equi）,meanwhile providing the foundation for prevention and diagnosis of equine theileriosis in this study,primers for merozoite surface protein antigen 1（EMA1） gene of T.equi based on the USDA strain sequence in the GenBank（AB043618） were designed to amplified the EMA1 gene of Zhaoyuan strain.Then the gene was cloned into the pMD19-T vector,sequenced and analyzed.A recombinant expression plasmid pET-30a-EMA1 was constructed by subcloning the cloned EMA1 gene into the linearized pET-30a vector.Then the plasmid pET-30a-EMA1 was expressed in Escherichia coli（BL21）.In result,the gene was 819 bp in size and encoded 272 amino acids.Comparison with 11 known amino acid sequence in the GenBank showed that the gene had the closest relationship with Zhaoyuan T.equi,followed by T.equi（Babesia equi）,Theileria annulata,and T.taurotragi,but distantly related with T.oriental,and T.sergenti.SDS-PAGE analysis showed that the expressed fusion protein was 38 ku in molecular mass.The protein could be recognized by T.equi positive serum in Western-blot.In conclusion,EMA1 was a good antigen of T.equi and had significant importance in taxonomy,epidemiological investigation,diagnosis and study of the candidate vaccine.