猪肺炎支原体DnaK基因的表达及ELISA方法建立  

Mycoplasma hyopneumoniae DnaK Gene and ELISA Method

作　　者：刘茂军[1] 丁志勇[1] 刘冬霞[1] 杜海霞[1] 缪芬芳[1] 甘源[1] 孔猛[1] 冯志新[1] 熊祺琰[1] 白方方[1] 杜改梅[2] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所.农业部兽用生物制品工程技术重点实验室.国家兽用生物制品工程技术研究中心,江苏南京210014 [2]金陵科技学院动物科学与技术学院,江苏南京210038

出　　处：《金陵科技学院学报》2011年第4期79-84,共6页Journal of Jinling Institute of Technology

基　　金：江苏省农业科技自主创新资金(cx(10)215);国家农业科技成果转化资金项目(2009GB2C100129)

摘　　要：猪肺炎支原体(Mhp)是猪气喘病的主要病原体。利用Mhp NJ株的DnaK基因通过SOE-PC(splicing withoverlap extension PCR)R扩增和突变,获得了目的片段并插入表达载体pET-28a(+)中,然后转入宿主菌BL21(DE3)。重组质粒经1 mmol/L IPTG在37°C温度下诱导5 h,目的蛋白可达到总蛋白的14.1%。Western Blotting证明表达的重组蛋白具有猪肺炎支原体反应原性,用该重组蛋白建立的ELISA方法可检测到猪肺炎支原体抗体。此为该病基因工程疫苗抗原的筛选和ELISA诊断试剂盒的研制奠定基础。Mycoplasma hyopneumoniae （Mhp） is the etiologic agent of mycoplasma pneumonia in swine. In this study the DnaK gene of strain NJ is amplified by gene splicing with overlap extension PCR using a pair of inside-mutation primers. The PCR product is inserted into expression plasmid pET-28a （＋）. The recombinant plasmid is transformed into competent cells of BL21（DE3） and it contains correct object fragments by sequencing. It is induced to express by 1 mmol/L IPTG at 37 ~C for 5 hours. The dose of the recombinant protein is nearly 14. 1~ of the total product. The expression product of interest and its biological activities are characterized with Western blotting and ELISA analysis. This study provides the basis for diagnose agent and future preparation of new vaccine a^ainst Mvconlasma hvonneurnnniae.

关 键 词：猪肺炎支原体 DNAK 重组蛋白 活性鉴定 

分 类 号：S852.62[农业科学—基础兽医学]