环形泰勒虫TA13815蛋白的原核表达及反应原性分析

Prokaryotic expression and reactivity analysis of TA13815 protein of Theileria annulata

为研究环形泰勒虫(Theileria annulata)TA13815蛋白功能,对环形泰勒虫虫体蛋白TA13815基因的核苷酸序列及编码蛋白的氨基酸序列进行生物信息学分析,构建pET30a-TA13815重组表达载体,并对其进行原核表达及反应原性分析。SDS-PAGE结果显示,蛋白的分子质量大约为29.663 ku,主要以包涵体形式表达,条带位置显示蛋白的分子质量比预期大,蛋白可能存在翻译后修饰。通过qPCR分析环形泰勒虫3个不同发育阶段,结果显示TA13815 mRNA在3个阶段均有表达,且在裂殖体阶段表达量最高。本试验成功表达了环形泰勒虫TA13815重组蛋白,该重组蛋白有较好的反应原性,且能与抗His标签蛋白的单克隆抗体和环形泰勒虫阳性血清发生特异性反应,而与健康牛血清无交叉反应。TA13815蛋白与东方泰勒虫的核苷酸和蛋白同源性均小于68%,表明该重组蛋白可作为间接ELISA诊断方法的备选抗原。

In order to study the function of Theileria annulata TA13815 protein,bioinfoiinatics analysis of the nucleotide sequence o£the T.annulata protein TA13815 gene and the amino acid sequence o£the encoded protein was conducted,the pET30a-TAI3815 recombinant expression vector was constructed,and its prokaryotic expression and reactogenicity analysis was carried out.The results analyzed by SDS-PAGE show that the molecular weight of the protein was about 29.663 ku,mainly expressed in the fonn of inclusion bodies.The band position showed that the molecular weight of the protein was larger than expected,and the protein might have been post-translational modified.The three different developmental stages of Theileria annulata analyzed by qPCR showed that TA13815 mRNA was expressed in all three stages,with the highest expression at the merozoite stage.In this study,the recombinant protein TA13815 was successfully expressed.It had good reactogenicity and could specifically react with monoclonal antibody against His-tagged protein and positive serum of Theileria annulata,but had no cross reaction with healthy bovine serum.The nucleotide and protein homology of TA13815 protein with T.orientalis was less than 68%,indicating that the recombinant protein could be used as an alternative antigen for indirect ELISA diagnostic methods.