多杀性巴氏杆菌prfA基因的序列分析与原核表达  

Sequence analysis and prokaryotic expression of prfAgene of Pasteurella multocida

作　　者：李影[1,2] 郭东春[2] 王淑亚[2,3] 张爱芹[2,4] 胡晓亮[1,2] 王雅静[2,3] 刘家森[2] 姜骞[2] 曲娟娟[1] 曲连东[2] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [3]东北农业大学动物医学院,黑龙江哈尔滨150030 [4]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国兽医科学》2014年第3期293-297,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31302109);国家科技支撑计划项目(2013BAK11B01)

摘　　要：为探索多杀性巴氏杆菌的致病机理，采用PCR方法分别扩增了家兔源和禽源多杀性巴氏杆菌C51—17、Pm898、Pm8916、Pm901、Pr09616、PmZM、PmQin菌株的prfA基因，并对其进行序列分析。将C51—17株的prfA基因克隆到质粒pET30a中，将重组表达载体pET30a—prfA转化到大肠杆菌BL21（DE3）pLysS感受态细胞中，经IPTG诱导表达，进行SDS—PAGE分析和Western—blot分析。结果显示，这7株菌株与GenBank登录的3株多杀性巴氏杆菌之间prfA基因在核苷酸水平上的同源性为95．6％～100％，在氨基酸水平上的同源性为98．9％～100％。诱导表达的重组蛋白PrfA的分子质量约为45．6ku，与预期的大小一致，以可溶性形式表达。重组蛋白PrfA可与家兔抗多杀性巴氏杆菌阳性血清反应，具有良好的反应原性。To explore the pathogenesis of Pasteurella multocida, the prfA gene was amplified by PCR from the genomic DNA of P. multocida strains Pm898, Pm8916, Ping01, Pm9616, PmZM, PmQin and CS1- 17,and the prfa gene of C51-17 strain was cloned into the prokaryotic expression plasmid pET30a. Then the recombinant plasmid pET-prfA was transformed into Escherichia coli BL21 （DE3） pLysS and induced by IPTG. The result of sequence analysis showed that the nucleotide homology of prfA gene was from 95.6% to 100% ,and the amino acid homology was from 98.9% to 100% between the 7 strains and other 3 strains of P. rnultocida retrieved from GenBank. SDS-PAGE analysis showed that the recombinant protein was about 45. 6 ku and present in the culture supernatant. Western-blot analysis indicated that the ex- pressed PrfA protein was recognized by positive serum against P. multocida.

关 键 词：多杀性巴氏杆菌 prfA基因 序列分析 原核表达 

分 类 号：S852.612[农业科学—基础兽医学]