布鲁氏菌L7/L12蛋白的基因克隆与原核表达

Gene Cloning and Prokaryotic Expression of L7/L12 Protein of Brucella

本研究对羊布鲁氏菌L7/L12蛋白进行了表达和纯化。首先从布鲁氏菌M5基因组中克隆L7/L12目的基因片段,连接至pMD-19T载体,转化入E.coli DH5α感受态细胞,PCR鉴定及测序鉴定正确后对其进行双酶切,构建重组质粒pGEX-6P-1-L7/L12并利用E.coli BL21(DE3)进行诱导表达。羊布鲁氏菌L7/L12基因片段大小为375 bp。SDS-PAGE检测蛋白大小为13 kD,与预测值相符。Western blotting方法检测其免疫学特性。实验结果表明,成功构建了pGEX-6P-1-L7/L12原核表达载体,并在大肠杆菌中成功表达了L7/L12重组蛋白,Western blotting法检测其具有免疫反应。本实验为下一步研究蛋白功能及布鲁氏菌新型疫苗的研制提供了实验基础。

In this research, the brucella protein L7/L12 gene was expressed and purified. First, the fragment of target gene L7/L12 was cloned from Brucella M5 genome, and then connected to p MD-19 T vector, and furtherly transformed into E. coli DH5 competent cells. After PCR identification and sequencing validation, the recombinant plasmid p GEX-6 P-1-L7/L12 was constructed and induced for expression in E. coli BL21(DE3). The fragment size of Brucella L7/L12 gene is 375 bp in length, and the size of encoding protein detected by SDS-PAGE is 13 k D, which is consistent with the predicted size. The immunological characteristics were detected by Western blotting. The results proved that the prokaryotic expression vector p GEX-6 p-1-L7/L12 was successfully constructed, and the recombinant protein of L7/L12 gene was successfully expressed in E. coli, and its immunological response was detected by Western blotting. This experiment provides an experimental basis for the further study of protein function and the development of noval vaccine for Brucella.