牛型布鲁氏菌A19株外膜蛋白OMP2b的表达及鉴定

Expression and identification of outer membrane protein 2b(OMP2b) of Brucella abortus strain A19

根据GenBank中的牛型布鲁氏菌基因序列,设计了1对特异性引物,以A19菌株基因组DNA为模板,通过PCR扩增出外膜蛋白OMP2b的全长基因,将OMP2b经T-A克隆后进行了序列测定和分析。结果表明,OMP2b全长1 128bp,编码376个氨基酸。将OMP2b定向克隆至表达载体pCold TF中,构建重组表达质粒pCold TF-OMP2b,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白His-OMP2b。SDS-PAGE分析结果显示,His-OMP2b约为93ku,与预期大小一致,表明His-OMP2b表达成功;经Wes-tern-blot分析,His-OMP2b能与牛型布鲁氏菌A19株全菌兔抗血清发生特异性反应,表明His-OMP2b具有免疫反应性。

According to the gene sequence of Brucella abortus available in GenBank,a pair of primers was designed and used for PCR amplification of the complete OMP2b gene from B.abortus strain A19.The PCR product was cloned into pMD18-T vector and subsequently sequenced.In result,the OMP2b gene was 1 128 bp in length,encoding 376 amino acids.The gene was then cloned into the expression vector pCold TF to construct recombinant plasmid pCold TF-OMP2b.The fused protein His-OMP2b was expressed in Esc-herichia coli BL21（DE3） by IPTG induction and identified to be 93 ku in size by SDS-PAGE,and reacted with rabbit antiserum against B.abortus by Western-blot,indicating that the fusion protein His-OMP2b had immunogenicity.