羊种布鲁氏菌Omp10基因的克隆及原核表达

Cloning and Prokaryotic Expression of Omp10 Gene of Brucella melitensis

根据GenBank公布的羊布鲁氏菌(B.melitensis)M5-90株外膜蛋白(outer membrane protein,Omp)基因序列,设计1对引物,以其全基因组为模板,采用PCR技术对其进行扩增,得到381bp的目的片段,连接入pMD20-T载体,转化E.coli DH5α感受态细胞;测序正确后,构建pET-28a-Omp10原核表达质粒,再将该质粒转化入E.coli BL21(DE3),IPTG诱导表达融合蛋白His-Omp10,用SDS-PAGE和Western blotting进行分析。结果表明,成功构建了含Omp10基因的原核表达载体,并在E.coli BL21(DE3)中表达了Omp10基因,诱导得到的融合蛋白经鉴定与目的蛋白大小一致,证明Omp10得到成功表达。该试验为布鲁氏菌病的进一步研究奠定基础。

According to the Brucella melitensis vaccine strain M5-90 outer membrane protein 10(Omp10)genetic sequence in GenBank,apair of primers was designed and the PCR technology was used to amplify Omp10 with its whole genome as the template.The Omp10 fragment which was 381 bp was obtained.Insert it into the plasmid pMD20-T,and it was transformed into E.coli DH5α.Recombinant prokaryotic expression plasmid,pET-28a-Omp10 was constructed,and transformed into E.coli BL21(DE3),and the fusion protein His-Omp10 was induced and expressed by IPTG,and identified with SDS-PAGE and Western blotting to analysis.The results showed that the recombinant protein was expressed successfully,which laid a solid foundation to the study of the animal immunization test profoundly.