牛型布氏杆菌GroEL的克隆表达及其生物学特性研究  

CLONING,EXPRESSION AND BIOLOGICAL PROPERTIES OF GROEL GENE OF BRUCELLA ABROTUS

作　　者：韩先干[1] 何随彬[2] 胡青海[1] 丁铲[1] 于圣青[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]南京农业大学动物医学院,南京210095

出　　处：《中国动物传染病学报》2010年第5期27-31,共5页Chinese Journal of Animal Infectious Diseases

基　　金：国家"973"项目(2010CB530202);中央级科研院所公益性研究专项(2010JB16)

摘　　要：根据已发表的牛型布氏杆菌(Brucella abrotus)基因序列设计一对特异性引物,以B.abrotusA19株基因组为模板,通过PCR扩增GroEL基因,经T-A克隆后进行序列测定和分析。结果表明,GroEL基因全长1 641bp,编码546个氨基酸。将GroEL定向克隆至表达载体pET32a中,构建重组表达质粒pET32a-LGroEL,然后转化大肠杆菌BL21(DE3),在IPTG诱导下成功获得重组表达蛋白His-GroEL,大小为81 kDa。经Western blot检测,牛布氏杆菌阳性血清中有GroEL抗体存在,表明该蛋白具有免疫原性。对重组表达蛋白His-GroEL进行酶促活性检测,结果表明该蛋白在体外具有催化ATP水解的酶活性和促进乳酸脱氢酶(lactate dehydrogenase,LDH)复性的功能。The primer pairs were designed for the amplification of Brucella abrotus strain A19 GroEL gene according to published sequence in GenBank.The PCR product was cloned into pMD18-T vector and sequenced.The result showed that the full-length of GroEL was 1 641bp in length,encoding 546 amino acids.GroEL was then cloned into the expression vector pET32a to construct recombinant plasmid pET32a-GroEL.The fused protein His-GroEL was expressed in E.coli BL21（DE3） via IPTG induction and identified as 81kDa in size.The antibodies to His-GroEL were detected in antiserum against B.abortus by SDS-PAGE and Western blot.In addition,GroEL was able to assist in vitro refolding of denatured L-lactate dehydrogenase（LDH） and performed ATPase activity.

关 键 词：布氏杆菌 GROEL 表达 酶活检测 

分 类 号：S852.614[农业科学—基础兽医学]