环形泰勒虫SPAG蛋白的原核表达及反应原性分析

根据GenBank中公布的环形泰勒虫子孢子表面抗原(SPAG)基因序列,结合生物信息学分析,对SPAG蛋白跨膜区和抗原表位进行分析。选取基因片段设计表达引物,从环形泰勒虫不同虫株的SPAG基因的保守区扩增目的片段,并构建SPAG-pET-30a重组表达载体,在大肠杆菌BL21(DE3)表达系统中进行表达,并对SPAG重组蛋白的反应原性进行分析。结果显示,获得的SPAG基因片段长度为882 bp,重组蛋白分子质量约为42 ku。反应原性分析显示,该重组蛋白可与环形泰勒虫阳性血清发生特异性反应,与中华泰勒虫、瑟氏泰勒虫、牛巴贝斯虫、双芽巴贝斯虫的阳性血清无交叉反应。本研究结果表明,SPAG蛋白有较好的反应原性和种特异性,可作为检测环形泰勒虫的候选抗原建立相应血清学诊断方法。

Prokaryotic expression and reactionogenicity analysis of recombinantTheileria annulata SPAG protein

The Theileria annulata sporozoite surface antigen(SPAG)gene sequence was obtained from GenBank database,the transmembrane region and the epitope of this gene were predicted via bioinformatic method.A pair of primers which designed from the conserved fragment ofSPAG gene,was used to amplify the target gene sequence from different isolates of T.annulata.The amplified gene was cloned into vector pET-30a(+)and expressed in Escherichia coli BL21(DE3)system.The purified recombinant SPAG protein was then used for reactionogenicity analysis.From the results,the amplified SPAG gene was 882 bp in length and molecular weight of the recombinant protein was 42 ku.Results of reactionogenicity analysis showed that the recombinant SPAG protein was specifically reacted with T.annulata positive serum,but no reactions with the positive sera for T.sinensis,T.sergenti,Babesia bovis,and Babesia bigemina.The present results indicated that the SPAG protein has good reactionogenicity and species specificity,and could be used as a candidate antigen to establish serological diagnostic method for T.annulata.