胸膜肺炎放线杆菌ApxIA N端基因的克隆与原核表达  

Cloning and Prokaryotic Expression of ApxIA Gene of Actinobacillus pleuropneumoniae

作　　者：吴笳笛[1] 苏禹刚[1] 费东亮[1] 

机构地区：[1]辽宁医学院畜牧兽医学院,辽宁锦州121000

出　　处：《安徽农业科学》2009年第6期2419-2421,共3页Journal of Anhui Agricultural Sciences

摘　　要：[目的]为猪接触传染性胸膜肺炎的诊断和基因工程疫苗的研制提供理论依据。[方法]以胸膜肺炎放线杆菌(APP)血清10型菌株为材料,采用PCR技术对其ApxIA的N端基因进行扩增,将扩增产物转入克隆载体和表达载体中进行克隆和表达,用重组表达质粒转化大肠杆菌BL21(DE3),利用免疫印迹分析鉴定ApxIAN端基因表达产物的免疫活性。[结果]ApxIA N端基因PCR扩增产物与标准10型ApxIAgene(D16582)的同源性为99.7%。NcoI和HindIII双酶切鉴定结果表明,目的基因已成功转入原核表达载体pET-32a中。含重组质粒的大肠杆菌BL21经IPTG诱导后表达了相对分子量为779 kDa的目的蛋白,该蛋白分子量与标准ApxIA蛋白(Marker)相同。[结论]SDS-PAGE电泳和免疫印迹检测结果表明,ApxIAN端基因在表达载体中得到高效表达,且表达产物具有免疫活性。[ Objective ] The study was to provide the theoretical basis for diagnosis on pig contact infectious pleuropneumoniae and development of genetic engineering vaccine. [ Method ] With 10 type strain of Actinobacillus pleuropneumoniae （APP） serum as the material, PCR technique was taken to amplify N-end gene of ApxIA, and the amplified products was transferred into cloning and expression vector to clone and express And the recombinant expression plasmid was taken to transform Escherichia coli BL21 （ DE3 ）, the immune activity of expression products of ApxIA gene was analyzed and identified by western blot. [ Result] The homology of PCR amplified products ofApxIA gene and standard 10 tape ApxIA gene（D16582） was 99.7%. The identification results of Nco Ⅰ and Hind Ⅲ double enzyme digestion indicated that the target gene was successfully transformed into prokaryotic expression vector pET-32a. After induction by IPTG, the Escherichia coli BL21 including recombinant plasmid expressed target protein which relative molecular weight was 779 kDa, and the molecular weight of the protein was the same with that of stander ApxIA protein（Marker）. [ Conclusion] The result of SDS-PAGE electrophoresis and western blot indicated that the N-end gene of ApxlA had high level expression in expression vector, and the expressed products had immune activity.

关 键 词：胸膜肺炎放线杆菌 毒素IA 克隆 检测 

分 类 号：S188[农业科学—农业基础科学]