牛巴贝斯虫棒状体相关蛋白1C端的克隆与表达

Cloning and expression of C terminal for rhoptryassociated protein 1 of Babesia bovis

用生物信息学方法,对牛巴贝斯虫棒状体相关蛋白(rhoptry-associated protein-1,RAP-1)的编码基因进行分析,并与其他巴贝斯虫和泰勒虫对应序列进行比对,以筛选出抗原性、特异性良好的RAP-1基因C端区域,设计特异引物,对基因进行克隆和原核表达,并应用Western-blot对重组蛋白的反应原性与特异性进行了分析。结果表明,目的蛋白的分子质量为51ku,可溶性表达量为1.51mg/mL,只与牛巴贝斯虫阳性血清发生特异性反应,而与其他巴贝斯虫、泰勒虫和无浆体阳性血清无交叉反应,预期可作为ELISA诊断方法的候选抗原,为牛巴贝斯虫病诊断方法的建立奠定了基础。

A C-terminal fragment with high antigenicity and specificity was screened from rhoptryassociated protein （RAP-l） of Babes＆ boris using bioinformatics analysis and sequence alignment with those of other Babesia and Theileria. The 3＇-terminal fragment of the RAP-1 gene was cloned and expressed in prokaryotic express system. The reactogenicity and specificity of the recombinant protein were evaluated by Western-blot. The results showed that the fusion protein had 51 ku and expression output as soluble protein was 1.51 mg/mL. Based on Western-blot analysis,the recombinant protein had strong reaction with the positive sera from B. boris infected cattle but no cross-reaction with positive sera against other Babesia,Theileria and Anaplasma species. It suggests the recombinant protein RAP-1 will be used as a candidate of diagnostic antigen to develop an ELISA technique. This study will be informative for developing serodiagnostic methods for B. boris infection in the future.