弓形虫PRU株BSR4基因的体外扩增及生物信息学分析

Amplification and bioinformatics analysis of Toxoplasma gondii BSR4 gene

目的:克隆弓形虫Prugniaud(PRU)株表面抗原BSR4基因,并进行序列测定和生物信息学分析。方法:根据BSR4基因已知序列(ME49株)设计合成一对引物,应用PCR技术从弓形虫PRU株基因组DNA中扩增BSR4基因,克隆入pET28a(+)载体并进行序列测定。用生物信息学方法对BSR4的理化性质、结构和功能进行预测。结果:得到弓形虫PRU株BSR4基因片段,测序结果表明,获得BSR4基因片段约1 194 bp,编码398个氨基酸。同源性分析显示,弓形虫PRU株和ME49株基因序列同源性为100%,BSR4相对分子量为42 344.75,有2个功能结构域,氨基酸序列的前40位为信号肽序列。N端为信号肽,C端为疏水序列,预测它为糖基磷脂酰肌醇固着蛋白,存在18个潜在抗原表位及2个保守功能区域。结论:成功获得弓形虫PRU株BSR4基因序列。

Objective：To clone and identify the surface antigen BSR4 of Toxoplasma gondii（T.gondii） PRU strain,to match the homology of BSR4 among T.gondii PRU and ME49 strains,and to analyze the feature by bioinformatics. Methods：DNA of strain PRU of T.gondii was extracted,and a pair of specific primers were designed from sequence of T.gondii ME49 bradyzoite surface antigen BSR4 and used to amplify the BSR4 of T.gondii PRU strain by PCR,then cloned into vector pET28a（＋） and sequencing.Predict the physical and chemical nature,structure and function of BSR4. Results：The target gene was amplified by PCR.The sequencing result showed that the fragment was 1 194 bp which encoded 398 amino acid.The deduce amino acid sequcence of PRU strain BSR4 gene showed a 100% homology with that of ME49 strain.The predicted BSR4 protein molecular weight is 42 344.75 Dalton which can form two functional domains with a signal peptide location before 40 amino acids.The N-terminal of BSR4 was a signal peptide and the C-terminal was a hydrophobic region which suggested it was a GPI-anchored surface protein.There were 18 potential antigenic epitopes and 2 conserved domains in BSR4. Conclusions：The sequence of bradyzoite-specific antigen BSR4 from T.gondii PRU strain was successfully cloned.