鼠疫耶尔森氏菌caf1和pH6基因的原核表达  

Prokaryotic expression of caf1 and pH6 antigen gene of Yersinia pestis

作　　者：郭荣[1] 刘青[1] 古丽阿依.包开西 张渝疆[1] 

机构地区：[1]新疆维吾尔自治区疾病预防控制中心,乌鲁木齐830002

出　　处：《疾病预防控制通报》2016年第1期24-27,共4页Bulletin of Disease Control & Prevention(China)

基　　金：卫生行业科研专项项目(201202021)

摘　　要：目的利用DNA重组技术,在大肠杆菌中获得融合表达的鼠疫耶尔森氏菌caf1及p H6抗原基因。方法利用分子克隆技术克隆后,在原核系统中表达鼠疫菌重要功能蛋白caf1及p H6蛋白,根据云南玉龙菌株(D106004)全基因组序列设计引物,PCR扩增目的基因片段;采用p ET-32a(+)作为表达载体,通过双酶切和连接反应,将目的基因片段定向插入载体中,构建重组表达质粒;IPTG诱导,使重组质粒在其宿主菌E.coli BL21(DE3)中表达。结果根据酶切鉴定和PCR产物测序结果显示,目的基因caf1及p H6已成功连接到表达载体p ET-32a(+)上,SDS-PAGE结果显示表达产物的相对分子质量约为34.2 ku和35.5 ku,与理论分子量一致。结论成功克隆并构建了p ET32a-caf1及p ET32a-p H6重组基因原核表达系统,为鼠疫潜在诊断靶点及新型疫苗选择的可能性奠定了基础。Objective To obtain caf1 and p H6 gene of Yersinia pestis in vitro by applying techniques of DNA recombination. Methods We cloned important functional protein of Y. pestis, namely caf1 and p H6 by the technique of molecular cloning, in prokaryotic systems for expression. According to complete genome sequence of Y. pestis strain D106004,detection primers were designed. The target DNA fragments were amplificated by polymerase chain reaction. Plasmid DNA named p ET-32a（＋） acted as expression vector. After digested by two different restriction enzymes and linked by T4 DNA ligase, the PCR products were cloned into p ET-32a（＋） in correct direction. The reconstructed plasmid was transformed into E. coli BL21 then. Fusion proteins expressed in E. coli BL21 after inducted by IPTG. Results According to the results of enzyme digestion and PCR product sequencing, objective gene caf1 and p H6 have been successfully connected to the expression vector p ET-32a（＋）,and SDS-PAGE results showed that the relative molecular weight of the expressed product was about 34.2 ku and 35.5 ku, and the molecular weight was consistent with the theory. Conclusions The prokaryotic expression system of p ET 32a-caf1 and p ET 32a-p H6 recombinant gene is successfully cloned and constructed for the plague potential diagnostic targets and the choice of new vaccines

关 键 词：鼠疫耶尔森氏菌 抗原基因 caf1 p H6 克隆表达 

分 类 号：R378.61[医药卫生—病原生物学]