布鲁氏菌BAB-RS25305蛋白对细胞炎症因子表达的影响

Effect of Brucella BAB-RS25305 Protein on Expressions of Inflammatory Cytokines

旨在对布鲁氏菌BAB-RS25305基因进行原核表达以及纯化,并探究其对细胞炎症细胞因子表达的影响。根据GenBank中所提供的BAB-RS25305基因序列(登录号:NC-007618.1)设计1对特异性引物,通过PCR扩增出BAB-RS25305基因片段并连接至PMD19-T载体,将构建好的PMD19-T-BAB-RS25305重组质粒转化至E.coli DH5α感受态细胞,提取质粒经HindⅢ、EcoRⅠ双酶切后将产物连接至pET-32a载体,然后转入E.coli BL21感受态细胞,用IPTG诱导表达并且纯化重组蛋白BAB-RS25305,将纯化后的蛋白以终浓度为50μL/mL刺激小鼠巨噬细胞RAW264.7,Real-time PCR和Western blot检测炎症相关因子NLRP3、caspase-1的表达变化。结果显示,成功构建了布鲁氏菌BAB-RS25305表达载体,经IPTG诱导表达后在32 ku左右出现一条特异性条带,与预期大小一致。用BAB-RS25305重组蛋白刺激小鼠巨噬细胞24 h后,发现与PBS对照组相比,BAB-RS25305重组蛋白试验组能够显著提高NLRP3以及caspase-1的表达。说明布鲁氏菌BAB-RS25305基因能够触发宿主细胞发生炎症反应,为揭示布鲁氏菌的胞内生存以及致病机理提供了理论依据。

The study was aimed to expression and purification of Brucella BAB-RS25305 gene,and to explore its influence on expressions of inflammatory cytokines.A pair of specific primers were designed based on the sequence of BAB-RS25305 gene(Accession No.:NC-007618.1)in GenBank.The BAB-RS25305 gene fragment was amplified by PCR method and ligated into the PMD19T-vector.The recombinant plasmid pMD19-T-BAB-RS25305 was constructed and then transformed into E.coli DH5ɑcompetent cells.The plasmid was identified by restriction enzyme digestion by HindⅢ,EcoRⅠ.The product was ligated into the pET-32a vector and transformed into E.coli BL21 competent cells.The recombinant protein BAB-RS25305 was expressed with IPTG induction and purified.The protein stimulated mouse macrophages RAW264.7 at a final concentration of 50μL/mL.Real-time PCR and Western blot were used to detect the expressions of inflammation-related factors NLRP3 and caspase-1.The results showed that the Brucella BAB-RS25305 gene expression vector was successfully constructed,and after IPTG induced expression,a specific band appeared at about 32 ku,which was consistent with the expected size.After stimulating mouse macrophages with BAB-RS25305 recombinant protein for 24 h,it was found that compared with the PBS control group,the BAB-RS25305 recombinant protein could significantly increase the expression of cytokine NLRP3 and caspase-1.It showed that the Brucella BAB-RS25305 gene can trigger the host cell's inflammatory response,which provides a theoretical basis for revealing the intracellular survival and pathogenic mechanism of Brucella.