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目的克隆牛布氏菌virB8基因并在大肠杆菌中进行表达。方法从牛布氏菌S19基因组DNA中PCR扩增virB8基因片段,插入pEASY-E1载体中,构建重组表达质粒pEASY-virB8,转化E.coli BL21(DE3),IPTG诱导表达。表达产物经HisTrapTMFF纯化后,进行SDS-PAGE分析和Western blot鉴定。结果扩增的virB8基因大小为720 bp;重组表达质粒pEASY-virB8经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约为30 000,表达量占菌体总蛋白的17.3%;纯化的重组蛋白纯度为85%,Lowry法测定蛋白浓度为1.52 mg/ml,可与鼠抗His单抗特异性结合。结论成功克隆了牛布氏菌virB8基因,并在大肠杆菌中表达了重组蛋白,为新型疫苗的研发及布氏菌鉴别诊断方法的建立提供了有效的候选抗原。
Objective To clone the virB8 gene of bovine brucella and express it in E. coli. Methods The virB8 gene fragment was amplified by PCR from S. bovis S19 genomic DNA and inserted into pEASY-E1 vector. The recombinant plasmid pEASY-virB8 was constructed and transformed into E. coli BL21 (DE3) for expression under induction of IPTG. The expressed product was purified by HisTrapTMFF and analyzed by SDS-PAGE and Western blot. Results The amplified virB8 gene was 720 bp in length. The recombinant plasmid pEASY-virB8 was confirmed by double enzyme digestion and sequencing. The relative molecular mass of the expressed recombinant protein was about 30 000, accounting for 17.3% of the total bacterial protein. Purified recombinant protein purity of 85%, Lowry method for the determination of protein concentration of 1.52 mg / ml, with mouse anti-His monoclonal antibody specific binding. Conclusion The virB8 gene was successfully cloned and the recombinant protein was expressed in E. coli. It provided an effective candidate antigen for the development of novel vaccines and the establishment of differential diagnosis of Brucella.