致羔羊脑炎粪肠球菌XJ05 Pfs基因的克隆、功能序列分析及原核表达  

Cloning,Functional Sequence Analysis and Prokaryotic Expression of Pfs Gene from Enterococcus faecalis XJ05 Inducing Lamb Encephalitis

作　　者：郑婷婷[1] 齐亚银[1] 王熙楚[1] 孔科委 周霞[1] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832000

出　　处：《西北农业学报》2015年第12期30-34,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31160507;31260610);人力资源与社会保障部出国留学人员科研启动项目(2010LX007);大学生创新创业训练项目(201210759021)~~

摘　　要：采用PCR技术从致羔羊脑炎粪肠球菌XJ05基因组中扩增得到甲硫腺苷/S-腺苷高半胱氨酸核苷酶(Pfs)基因的全序列,对其功能序列进行分析,同时与其他细菌相应序列及编码的氨基酸进行同源性分析,构建该基因的原核表达载体,对表达蛋白进行反应原性分析。结果显示:XJ05的Pfs基因全长696bp,与XJ01、XJ08、XJ11,与屎肠球菌TX 2466之间的同源性在99.1%以上,其中,XJ05与V583为100%,且关键的功能序列相同,与肺炎链球菌同源性在55.8%以上、霍乱弧菌同源性在55.0%以上;pfs基因编码231个氨基酸,XJ05、XJ01、XJ08、XJ11与肺炎链球菌、霍乱弧菌、粪肠球菌V583、屎肠球菌TX 2466的同源性在86.43%以上;构建的重组质粒经诱导后表达蛋白分子量为28ku,Western blot检测显示特异性条带。结果表明,表达的融合蛋白具有良好的反应原性。The Pfs gene, which functions in lamb encephalitis, was amplified by PCR technology from Enterococcus faecalis XJ05, then sequenced and conducted BLAST with the corresponding sequences of DNA fragment and amino acid in GenBank. Moreover, the prokaryotic expression vector was constructed and the recombinant proteins were analyzed by SDS--PAGE and Western blot. The results showed that the full--length sequence of Pfs gene is 696 bp, with the homology at least 99. 1 % compared with E. faecalis XJ01, XJ08 ,XJ11, and E. faeciurn TX2466, 55.8% with Strep pneumoniae, and 55.0 % with Vibrio cholerae, and their important functional sequences are the same. The pfs gene encoding 231 amino acids, with the homology of at least 86.43% compared with XJ05, XJ01, XJ08 and XJ11, Strep pneumoniae, Vibrio cholerae, E. faecalis V583 and E. faecium TX2466. The expressed product of gene is 28 ku, Western blot showed a specific hand, which suggested a good immunogenicity of the recombinant protein.

关 键 词：粪肠球菌 Pfs基因 序列分析 原核表达 

分 类 号：S852.611[农业科学—基础兽医学]