摘要
目的研究在结核分枝杆菌感染过程中,ppe37蛋白引起的巨噬细胞免疫应答反应。方法利用本实验室已构建好的ppe37原核表达载体,将其转化到大肠杆菌E.coli BL21中,用IPTG诱导表达后,经SDS-PAGE与Western blot鉴定后,利用Ni-Agarose His标签蛋白纯化试剂盒对重组蛋白进行纯化并复性。利用SDS-PAGE和AlpHaImager 2200软件对纯化蛋白进行鉴定与分析。分别用浓度为100ng/mL、500ng/mL、5 000ng/mL的重组ppe37蛋白刺激RAW264.7巨噬细胞,24h、48h、72h后,用Real-time PCR检测RAW264.7巨噬细胞NF-κB、TNF-α、IL-6mRNA的表达量的变化。结果 SDSPAGE鉴定显示,重组ppe37蛋白获得了大量表达,其相对分子质量约为50kDa。经Western Blot验证,重组ppe37蛋白可与抗His单抗发生特异性反应,经AlpHaImager 2200软件薄层扫描分析,重组蛋白的纯度为96.2%,并成功的进行了蛋白复性。浓度分别为100ng/mL,500ng/mL,5 000ng/mL的重组ppe37蛋白刺激巨噬细胞,24h后Real-Time PCR检测结果显示与空白对照组相比NF-κB、TNF-αmRNA的表达没有影响(P>0.05),而IL-6mRNA的表达上调(P<0.05);48h、72h后NF-κB、TNF-α、IL-6mRNA的表达显著上调(P<0.05)。结论成功地制备并纯化了重组ppe37蛋白,重组ppe37蛋白能够活化巨噬细胞,激活细胞免疫反应,并促使巨噬细胞发生炎性反应。
The purpose of this study was to evaluate the immune response of macrophages by ppe37 protein in the process of human infection with Mycobacterium tuberculosis. The ppe37 prokaryotic expression vector was ready-built in our laboratory. After being transformed into E. coli BL21 and induced with IPTG, the recombinant ppe37 protein was identified by SDS-PAGE and Western Blot, and then it was purified by the nickel column purified protein kit and refolded. The purifie＇d protein was identified and analyzed by SDS PAGE and AlpHaImager 2200 software. The RAW264. 7 cells were infected by ppe37 protein with various concentrations respectively （100 ng/mL, 500 ng/mL, 5 000 ng/mL）. After culturing in humidified air atmosphere for 24 hours, 48 hours and 72 hours, the RAW264.7 cells were harvested for real-time PCR analysis of inflammatory cytokine gene expression including the NF-κB, TNF-α and IL-6. Results showed that the molecular weight of recombinant ppe37 protein was 50 kDa, and it was highly expressed in E. coli BL21 after induction with IPTG. Western-blotting analysis showed that the recombinant protein was specifically reacted with antihistidine monoclonal antibody. TLC scanning analysis of AlpHalmager 2200 software showed that its purification was 96.2%, and the recombinant ppe37 protein was successfully refolded. After 24 hours, compared with the control, the expression of NF-κB or TNF-α had no effect on cells infected with 100 ng/mL, 500 ng/mL and 5 000 ng/mL ppe37 protein （P〉0.05）, but IL-6 was up-regulated （P〈0.05）. After 48 hours and 72 hours, the expressions of NF-κB, TNF-α, and IL-6 were up-regulated in cells infected with various concentrations of ppe37 protein （P〈0.05）. The recombinant ppe37 protein was successfully produced and purified in this study and it＇s proved that the protein could activate macrophages and cellular immune response and promote the process of macrophage inflammatory reaction.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第8期803-807,共5页
Chinese Journal of Zoonoses
基金
国家自然科学基因(No.31160494)
2011年教育部“新世纪优秀人才支持计划”项目(No.NCET 11-11023)
宁夏高等学校科学技术研究项目(2010)联合资助~~
