田鼠巴贝斯虫棒状体蛋白相关因子1基因的原核表达及其特性研究

PROKARYOTIC EXPRESSION AND BIOINFORMATICS ANALYSIS ON RHOPTRY-ASSOCIATED PROTEIN INTERACTING FACTOR 1 GENE OF BABESIA MICROTI

本研究以田鼠巴贝斯虫基因组DNA为模板,扩增棒状体蛋白相关因子1(rhoptry-associated protein interacting factor 1,简称RAPIF1)基因,全长为771 bp,编码256 aa,编码蛋白的分子质量为29 kDa。利用多种数据库与生物信息学分析软件,对田鼠巴贝斯虫RAPIF1进行生物信息学分析,预测其亲水性/疏水性、跨膜区、信号肽、翻译后修饰位点、B淋巴细胞抗原表位,结果预示RAPIF1具有良好的亲水性。将RAPIF1的ORF插入表达载体pET-28a进行原核表达,重组蛋白rRAPIF1大小为37 kDa。制备rRAPIF1的多克隆抗体,Western blot结果显示,RAPIF1存在于虫体中,且rRAPIF1具有良好的反应原性,可有效区分田鼠巴贝斯虫阳性血清和阴性血清。本研究结果表明,RAPIF1预期可作为田鼠巴贝斯虫检测和疫苗制备的候选分子。

In this study, rhoptry-associated protein interacting factor 1 (RAPIF1) was cloned from Babesia microti (B.microti ) gDNA.The full length nucleotide of RAPIF1 was 771 bp, encoding 256 aa with a predicted molecular weight of 29 kDa. The RAPIF1 gene was analyzed by bioinformatics for characterization of its hydrophilicity/hydrophobicity, transmembrane domains, signal peptides, posttranslational modification sites and potential B cell antigen epitopes. The RAPIF1 gene was cloned into pET-28a and expressed in E.coli as a 37 kDa His-fusion protein (rRAPIF1). RAPIF1 existed in B. microti and recombinant RAPIF1 protein reacted with B. microti positive serum as demonstrated in Western blot. These results indicated that RAPIF1 could be a potential candidate for detection and vaccine development for B.microti.