摘要
利用PCR技术从单增李斯特氏菌中扩增出actA基因,将PCR产物纯化后克隆到pMD 18Tsimple vector中,成功构建出克隆载体pMD-18T/actA。以BamHⅠ和EcoRⅠ分别双酶切pMD-18T/ActA和表达载体pGEX-3X,将纯化的actA基因亚克隆到表达载体pGEX-3X。构建的重组表达载体pGEX-3X/ActA转化到E.coli BL21(DE3)中,IPTG诱导表达,表达产物进行SDS-PAGE分析,可见约120 ku外源蛋白带,Western blot分析表明该蛋白可与单增李斯特氏菌多克隆抗体发生特异性反应。该研究为ActA的生物学特性和功能的研究及诊断试剂的研制奠定了基础。
The gene encoding ActA was amplified from Listeria monocytogens by PCR. Cloning vector pMD-18T/ActA was successfully constructed by cloning the PCR product into pMD 18T simple vector, pMD-18T/ActA and pGEX-3X were digested by BamH I and EcoR I . The recombinant expression vector pGEX-3X/ActA was constructed by subcloning the actA gene into the expression vector pGEX-3X, and transformed into E. coli BL21 （DE3）. The bacteria were induced by IPTG and the lysates were analyzed by SDS-PAGE. The expressed fusion protein showed a molecular weight of 120 ku. The protein could react with polyclonal antibody against L. monocytogens in Western blot. The results provide a foundation for further studies on the biological characteristics of ActA and its role in listeria pathogenesis and for diagnosis agent.
出处
《畜牧与兽医》
北大核心
2008年第3期14-17,共4页
Animal Husbandry & Veterinary Medicine
