多杀性巴氏杆菌HN06株磷酸甘油酸变位酶gpmA基因的序列分析及其原核表达  

Sequence analysis and prokaryotic expression of gpmAgene encoding phosphoglycerate mutase of Pasteurella multocida strain HN06 originated from swine

作　　者：曾东柱 李慧[1] 孔汉金[1] 郭锐[1] 吴斌[1] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医科学》2015年第1期57-60,共4页Chinese Veterinary Science

摘　　要：为探索糖酵解关键酶磷酸甘油酸变位酶的功能,采用PCR方法从多杀性巴氏杆菌HN06株中扩增出了磷酸甘油酸变位酶基因gpmA,将其克隆到表达载体pET-30a(+)中,测序验证后转入表达菌株大肠杆菌BL21中进行IPTG诱导表达。结果显示,重组菌可表达出分子质量约为32ku的重组融合蛋白,在终浓度为20mmol/L IPTG诱导3h的情况下表达良好。表达的蛋白以可溶性蛋白的形式存在于菌体中。表达产物经Ni柱纯化后可得到纯度较高的目的蛋白。经过Western-blot分析,所纯化的蛋白能与抗多杀性巴氏杆菌HN06株阳性血清进行特异性的免疫印迹反应,证实表达的蛋白具有良好的反应原性。GpmAgene encoding phosphoglycerate mutase of Pasteurella multocida strain HN06 originated from swine was amplified by PCR and cloned into pET-30a(+)expression vector.After being sequenced,the recombinant plasmid was transformed into Escherichia coli BL21.The transformed bacteria were induced with IPTG and produced a recombinant protein of 32 ku in mass.In result,20mmol/L IPTG and 3hof induction time were the excellent conditions for the protein production and more pure proteins would be produced after Ni+-column purification.The purified protein could react with the positive serum with the antibody against strain HN06 in a Western-blot test,which showed that the expressed protein had strong reactionogenicity.

关 键 词：多杀性巴氏杆菌 磷酸甘油酸变位酶 gpmA基因 原核表达 纯化 免疫印迹 

分 类 号：S852.612[农业科学—基础兽医学]