布鲁氏菌外膜脂蛋白OMP10基因的克隆表达、蛋白纯化及免疫原性研究

Cloning,Expression,Purification and Immunogenicity Test of Brucella Lipoprotein OMP10

根据GenBank中的牛型布鲁氏杆菌OMP10序列,设计一对引物,以布鲁氏菌疫苗株S2全基因组DNA为模板扩增OMP10基因,将目的基因克隆入pEASY-T3,经测序正确后,与载体pET-32a(+)连接,构建重组表达质粒pET-32a/OMP10,转化E.coli BL21(DE3),用IPTG诱导表达融合蛋白pET-32a/OMP10,经SDS-PAGE及Western-blot分析鉴定后,采用Ni-NTA Spin Kit纯化目的蛋白,并免疫小鼠。结果表明,成功构建了含OMP10的表达载体,经多次对表达条件的优化,获得了纯度较高的目的蛋白,为进一步动物免疫试验及研究其免疫原性和抗感染保护作用奠定了基础。

To construct a prokaryotic expression vector for Brucella OMPIO, as well as express and puri- fy the recombinant protein, Brucella S2 genome sequence was extracted and used as temple for polymerase chain reaction to amplify OMPIO gene. Then the PCR product was cloned into the pEASY - T3 vector and subsequently sequenced. Then the gene was cloned into the expression vector pET - 32a （ ＋ ） to construct the recombinant plasmid pET- 32a/OMP10. The fused protein pET -32a/OMP10 was expressed by IPTG induc- tion and examined by SDS - PAGE and Western - blot, then purified by Ni - NTA Spin Kit. In this study, Brucella OMP10 was successfully cloned, the expression condition was optimized, the target protein was strongly expressed, and highly purified protein was obtained. This study laid a foundation for further research of animal examination, immunogenicity and protection of brucellosis.