布鲁氏菌外膜蛋白16基因的克隆及原核表达

为了成功克隆外膜蛋白16(outer membrane proteins 16, Omp16)基因并对其进行原核表达,试验根据GenBank中羊布鲁氏菌M5-90株外膜蛋白Omp16基因序列(登录号:JF918760.1)设计1对引物,从布鲁氏菌基因组中扩增出大小约为507 bp的目的基因片段,凝胶回收纯化目的片段,连接入pMD20-T质粒,转化E.coli DH5α并测序,测序正确后再亚克隆入pET-28a(+)表达载体,构建重组质粒pET-Omp16,转化入E.coli BL21(DE3),经IPTG诱导其表达,最后用Western blotting分析方法鉴定诱导得到的蛋白。结果表明,成功构建了pET-Omp16原核表达载体,并在E.coli BL21中表达了Omp16基因,诱导得到的蛋白经鉴定与目的蛋白大小一致,证明成功表达了目的基因。

Cloning and Prokaryotic Expression of Outer Membrane Proteins 16 Gene of Brucella melitensis

To successfully clone the outer membrane proteins 16 (Omp16) gene and make prokaryotic expression in E.coli, one pair of primers was designed according to B. melitensis M5-90 strain Omp16 gene sequence in GenBank, and then obtained Omp16 gene which was about 507 bp by PCR from the Brucella genome. After purifying, Omp16 gene was inserted into pMD20-T vector to construct recombinant plasmid pMD-Omp16. pMD-Omp16 transformed into E.coli DH5α and identified it by sequencing, then subcloned to vector pET-28a(+). The constructed recombinant plasmid pET-Omp16 was transformed into E.coli BL21(DE3) for expression under induction of IPTG. Lastly, the expression products of recombinant protein His-Omp16 was identified by Western blotting. The results showed that the prokaryotic expression vector was successfully constructed and expressed Omp16 gene in E.coli BL21.