摘要
PCR扩增单增李斯特氏菌ActA基因,纯化后克隆到pMD 18T simple vector中,以BamHI和EcoRI双酶切克隆载体pMD-18T/ActA,再将其亚克隆到表达载体pGEX-3X中,获得重组质粒pGEX-3X/ActA,转化E.coli BL21(DE3)细胞,IPTG诱导融合蛋白(GST-ActA)表达,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。试验采用Glutathione Sepharose 4B亲合层析纯化融合蛋白。对单增李斯特氏菌ActA的原核表达与纯化的研究为单增李斯特氏菌诊断试剂的研制及ActA的免疫原性研究奠定了基础。
The gene encoding ActA was amplified from Listeria monocytogens by PCR.The purified PCR product was cloned into pMD 18T simple vector.The pMD-18T/ActA was digested by BamHI and EcoRI double enzymes and the actA gene was subcloned into the expression vector pGEX-3X.The recombinant plasmid pGEX- 3X/ActA was transformed into E.coli BL21 （DE3）.The bacteria was induced by IPTG and its lysate was analyzed by SDS-PAGE and Western blot.The fusion protein can react with against Listeria monocytogens polyclonal antibody. The GST-tagged protein was purififed by affinity chromatograph.This work provides a foundation for further studies on preparation for diagnosis agent and the iminunogenicity of ActA .
出处
《现代畜牧兽医》
2008年第3期50-53,共4页
Modern Journal of Animal Husbandry and Veterinary Medicine
关键词
单增李斯特氏菌
ACTA
原核表达
蛋白纯化
Listeria monocytogens
ActA
prokaryotic expression
protein purification
