巴尔通体（Bartonella tribocorum）P26蛋白原核重组表达研究

Expression of Recombinant p26 Protein of Bartonella tribocorum

目的 构建巴尔通体表面蛋白p26基因的原核重组表达载体,表达和纯化重组表达蛋白P26,并鉴定其抗原性.方法 利用PCR方法从巴尔通体B.tribocorum厦门分离株的基因组DNA中扩增出p26蛋白基因,并将该基因的编码区克隆到pGEX-4T-1表达载体中,从而构建GST-p26融合蛋白原核重组表达载体.将表达载体转化大肠埃希菌（E.coli DH5α）,诱导表达GST-p26,并运用亲和层析技术纯化蛋白.通过蛋白质斑点印迹试验和间接ELISA法检验GST-p26是否具有抗原性.结果 成功构建了p26的原核表达载体,该表达载体可在E.coli DH5α中大量表达GST-p26,原核表达的GST-p26能够与感染动物血液样本发生特异性免疫反应.结论 原核重组表达的GST-p26可作为潜在的抗原,应用于巴尔通体的血清学检测.

Objective To generate prokaryotic expression vector for Bartonella surface protein p26 gene, express and purify the recombinant p26, and characterize its antigenicity. Methods p26 gene was amplified from the genomic DNA of B. tribocorum isolated from Xiamen region through PCR assay. The coding region of p26 was cloned into pGEX-4T-1 vector to generate the prokaryotic vector expressing recombinant fusion protein GST-p26. The expression vector was transformed into E. coli DH5ot to express GST-p26. GST-p26 was purified through affinity chromatography assay. The antigenicity of GST-p26 was investigated with Western-blot and indirect ELISA assay. Results We successfully generated the recombinant p26 protein prokaryotic expression vector. The expression vector was induced to produce GST-p26 efficiently. Western-blot experiment indicated that the expressed prokaryotic GST-p26 immunologically reacted with the blood samples from the infected animal. Conclusion The results demonstrated that the expressed recombinant GST-p26 is a potential antigen for serological detection of Bartonella.