奶牛乳腺炎源性大肠杆菌csgC基因的克隆及载体构建  

Cloning and construction of recombinant expression plasmid of dairy cattle mastitis Escherichia coli strain csgC gene

作　　者：王林锋[1,2] 杨宏军[2] 杨少华[2] 王长法[2] 高运东[2] 钟跻峰[2] 葛利江[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]山东省农业科学院奶牛研究中心,山东济南250100

出　　处：《中国兽医学报》2009年第4期441-444,共4页Chinese Journal of Veterinary Science

基　　金：国家“863”计划资助项目;山东省农业科学院重大成果培育基金资助项目(2006YCG012);山东省农业科学院高技术自主创新基金资助项目(2006YCX027)

摘　　要：根据GenBank上发表的curli菌毛csgC基因序列设计了1对特异性引物。从患乳腺炎的奶牛乳汁中分离出致病性大肠杆菌,经生物学鉴定后,提取全基因组DNA为模板,PCR扩增出csgC基因,连入pMD18-T克隆载体,测序,结果表明,扩增片段含有333个核苷酸,编码111个氨基酸的成熟蛋白,与已报道的大肠杆菌W3110的全基因组DNA中的csgC基因序列最相近,氨基酸序列同源性为99.7%。该基因与原核表达载体pET32a+相连,转入BL21(DE3)感受态细胞。挑选阳性菌落经PCR鉴定和酶切鉴定后表明成功构建原核表达载体pET32a+/csgC。One pair of specific primers to curli fibers csgC gene was designed and synthesized according to the published sequence in GenBank. Escherichia coli were isolated and identified from the milk of cows with mastitis,csgC gene was cloned from genomic DNA by PCR, and the PCR product was inserted into vector pMD18-T to construct plasmid pMD18-T/csgC,Sequence analysis showed that the amplified fragment was composed of 333 nucleotides,encoding a mature polypeptide with 111 amino acids, compared with the reported csgC gene of the whole genome DNA sequence in W3110, the highest amino acid identity was 99.7 % The products were inserted into the vector pET32a＋ ,and diverted into BL21（DE3） competent cells. The positive colony which were identified by digestion and PCR showed that the recombinant plasmid pET32a＋/csgC was constructed successfully. This study provides conditions for the research of pathogenic and immune mechanism and vaccine design.

关 键 词：大肠杆菌 curli菌毛 csgC基因 表达 

分 类 号：S852.61[农业科学—基础兽医学]