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PCR扩增布鲁菌rplL、groEL、bp26、omp25、p39、sod1基因,构建pET-28a(+)-rplL、pCold I-groEL、pCold I-bp26、pCold I-omp25、pCold I-p39、pCold I-sod1原核表达质粒,分别转化大肠埃希菌BL21(DE3)感受态细胞,经IPTG诱导后,SDS-PAGE和Western blot分析蛋白的表达情况,免疫亲和纯化目的蛋白。结果表明:PCR分别扩增出375、1 641、753、642、1 206和522bp的目的片段,重组菌诱导后分别表达大小为20、65、33、28、47和24ku的重组蛋白,与目的蛋白理论大小相符。Western blot结果显示6种重组蛋白均能与His单抗发生特异性反应,表明6种重组蛋白具有良好的免疫反应性,为进一步深入研究奠定了基础。
PCR was used to amplify the rplL, groEL, bp26, omp25, p39 and sod1 genes of Brucella, to construct pET-28a (+) - rplL, pCold I-groEL, pCold I-bp26, pCold I -omp25, pCold I-p39, pCold I-sod1 prokaryotic expression plasmid was transformed into Escherichia coli BL21 (DE3) competent cells induced by IPTG, SDS-PAGE and Western blot analysis of protein expression, immunoaffinity purification of the target protein. The results showed that the target fragments of 375, 641, 753, 642, 1 206 and 522 bp were amplified by PCR respectively. The recombinants expressed recombinant proteins of 20, 65, 33, 28, 47 and 24ku respectively, Protein theoretical size match. Western blot results showed that all six recombinant proteins reacted specifically with His monoclonal antibody, indicating that the six recombinant proteins have good immunoreactivity and laid the foundation for further study.