布鲁氏菌外膜蛋白2b基因的克隆、原核表达及蛋白生物信息学分析

Cloning,Prokaryotic Expression and Bioinformatics Analysis of Omp2b Gene of Brucella melitensi

本试验旨在克隆布鲁氏菌外膜蛋白2b(Omp2b)基因并进行原核表达和蛋白的生物信息学分析。根据布鲁氏菌M5-90株外膜蛋白Omp2b基因序列设计引物,以布鲁氏菌基因组为模板,通过PCR技术扩增得到Omp2b基因片段,回收纯化后,将此片段连接入pMD20-T质粒,将该重组质粒转化E.coli DH5α感受态细胞,挑取阳性克隆菌提取质粒后,送公司测序。将该片段亚克隆入pET28a载体,构建pET28a-Omp2b表达载体,转化E.coli BL21(DE3)菌株,IPTG诱导其表达,用SDS-PAGE和Western blotting分析鉴定此蛋白。运用DNAMAN、BioEdit等各种工具软件对Omp2b基因编码的氨基酸序列进行分析。结果显示,成功克隆了Omp2b基因,其开放阅读框为1041bp,编码347个氨基酸;构建了pET28a-Omp2b原核表达载体,并在E.coli BL21(DE3)中成功表达了Omp2b基因,表达蛋白约38ku;Omp2b蛋白二级结构中α-螺旋、伸展链、β-折叠和无规卷曲分别占20.17%、26.22%、5.76%和47.84%。

The study was aimed at cloning, expressing the gene of outer membrance protein 2b （Omp2b） and analyzing the bioinformatics of Omp2b, according to the nucleotide sequence of Omp2b gene of Brucella melitensis M5-90, a pair of primers was designed, and Omp2b gene fragment was amplified from Brucella genome by PCR. The fragment was identified and cloned into prokaryotic expression vector pMD20 T. After sequencing, Omp2h fragment was ligated into pET28a to construct the re- combinant expression plasmid pET28a-Omp2b, and then transformed into the E. coli BL21 （DE3） strains. IPTG was used to induce expression of Omp2b protein,and the protein was detected by SD^PAGE and Western blotting. DNAMAN and BioEdit softwares were used to analyze the sequence of amino acids encoded by Omp2b gene. The results showed that Omp2b gene was cloned successfully~ the ORF was 1041 bp, which encoded 347 amino acids; the expression vector pET28a-Omp2b was con structed; the molecular weight of the expression protein was about 38 ku; Omp2b protein secondary structure of alpha helix, extended strand, 13 turn and random coil were accounted for 20.17%, 26.22%, 5.76% and 47.84%, respectively.