鸭疫里默氏菌甲羟戊酸激酶基因的重组表达与免疫保护效果研究

Expression and Immunogenicity Study of Mevalonate Kinase Gene of Riemerella anatipestifer

在从鸭疫里默氏菌(RA)基因组文库筛选毒力基因的过程中,作者获得了编码RA甲羟戊酸激酶的基因(RAMVA),将该基因片段提交GenBank,收录号为GQ398751。测序分析结果显示其开放阅读框长492 bp,共编码163个氨基酸。根据序列信息设计引物,用PCR方法从RA中扩增出RAMVA的编码区并与表达质粒pMAL-C2X连接,构建重组质粒pMAL-RAMVA。经限制性酶切和序列测序鉴定序列正确无误后将pMAL-RAMVA转化至E.coliBL21(DE3)构建重组表达菌E.coliBL21/pMAL-C2X-RAMVA。用IPTG诱导重组菌成功诱导RAMVA的表达,通过SDS-PAGE分析表达产物,表达量占全菌总蛋白的15.6%,且重组表达的甲羟戊酸激酶在大肠杆菌中表达时以可溶性状态存在。雏鸭免疫试验表明,接种重组表达菌E.coliBL21/pMAL-C2X-RAMVA2周后,可产生对RA致死量攻击达42.3%的免疫保护率。

Mevalonate Kinase gene（RAMVA） was obtained when virulent genes of Riemerella anatipestifer were screened from DNA library by specific antibody as probes.The DNA fragment named DQ398751 had been record by GenBank.The length of open reading frame of RAMVA was 492 bp and encoding 163 amino acids.In order to express the gene,the digested PCR product of RAMVA was ligated with digested vector pMAL-C2X,and transformed into host bacteria E.coli BL21（DE3）.Finally,the gene was successfully expressed in the bacteria by induction of IPTG,the content of the recombinant protein was about 15.6% of the whole bacteria proteins,and the recombinant mevalonate kinase was in a soluble state.Ducklings′ immune tests showed that two weeks post inoculation of the recombinant bacteria E.coli BL21/pMAL-C2X-RAMVA,the protection rate to Riemerella anatipestifer infection was 42.3%.