布鲁氏菌OMP22蛋白的原核表达、纯化及其对巨噬细胞炎性反应因子mRNA表达的影响观察

Prokaryotic expression and purification of Brucella OMP22 protein and its effect on macrophage expression of mRNA of inflammatory response factors

目的原核表达布氏杆菌OMP22蛋白并观察其对巨噬细胞炎性因子mRNA表达的影响。方法在GenBank中查找牛型布鲁氏菌Omp22基因序列并进行引物设计,以其疫苗的全基因组DNA为模板PCR扩增Omp22基因,扩增产物克隆入PEASY-T3载体后测序,测序正确的基因片段与载体pET-32a(+)连接,构建重组表达质粒pET-32a-Omp22,转化感受态细胞BL21(DE3)后,通过IPTG诱导表达融合蛋白OMP22。用胶回收纯化的目的蛋白作用于小鼠巨噬细胞RAW264.7并检测其相关炎性因子mRNA水平。结果结果表明成功构建了布鲁氏菌Omp22蛋白的表达载体,转化DE3后经IPTG诱导表达约35×10~3的目的蛋白,与预期大小相符。Western blot检测该蛋白能被His抗体识别。用纯化的OMP22刺激小鼠巨噬细胞,其IL-1β、IL-6、IL-8、TNF-α等炎性因子表达水平显著降低。结论 OMP22蛋白能降低巨噬细胞的炎性反应表达,为布病的进一步研究提供了理论依据。

Objectives To express the Brucella OMP22 protein in prokaryotic cells and top observe its effect on the expression of macrophage inflammatory factors.Methods GenBank was searched for the gene sequence of Brucella bovis OMP22,and a primer was designed.Genomic DNA of the vaccine strain was used as a template to amplify the OMP22 gene.The product of amplification was cloned into a PEASY-T3 vector and sequenced.The gene fragment was ligated into a pET-32 a(+) vector,and the recombinant expression plasmid pET-32 a-OMP22 was constructed.The plasmid was transformed into BL21(DE3) competent cells.Expression of the fusion protein OMP22 was induced with IPTG,and murine macrophages were treated with the purified target protein.RAW264.7 phagocytes were tested for their level of expression of mRNA of related inflammatory factors.Results An expression vector for Brucella OMP22 protein was successfully constructed.After a plasmid was transformed into BL21(DE3) cells,expression of about 35×103 of the target protein was induced with IPTG.Results were consistent with expectations.The protein was specifically bound by His-OMP22 antibodies according to Western blotting.Mouse macrophages were stimulated with purified OMP22 protein to decrease the levels of expression of inflammatory factors such as IL-1β,IL-6,IL-8,and TNF-α.Conclusion OMP22 protein reduced the expression of an inflammatory response in macrophages.This finding provides a theoretical basis for further research on brucellosis.