摘要
[目的]探讨鸟分枝杆菌PPE25在调控人源THP-1巨噬细胞免疫应答反应中的作用及机制。[方法]通过基因扩增、载体构建、诱导表达、分离纯化等过程制备鸟分枝杆菌重组PPE25蛋白;将重组PPE25蛋白作用于THP-1巨噬细胞后,利用流式细胞术检测巨噬细胞凋亡率;运用ELISA方法检测细胞上清中TNF-α的浓度;采取Western Blotting方法检测Bax、Bcl-2、TLR2、TLR4的蛋白表达水平;采用q PCR法检测IL-6、IL-12的mRNA表达水平。[结果]利用大肠杆菌表达系统成功制备重组PPE25蛋白;重组PPE25蛋白作用巨噬细胞后,细胞凋亡率升高(P <0.05),细胞中Bax蛋白表达水平升高(P <0.05),而Bcl-2蛋白表达水平降低(P <0.05);另外,细胞中TLR2、TLR4蛋白表达水平上升(P <0.05),TNF-α含量升高(P <0.05),IL-6及IL-12表达水平升高(P <0.05)。[结论]鸟分枝杆菌PPE25蛋白可以促进人源THP-1巨噬细胞凋亡,并可以通过激活巨噬细胞TLR2或TLR4信号途径促进细胞炎症因子IL-6及IL-12的分泌,从而诱导机体产生免疫应答,抵御鸟分枝杆菌感染。
[Objective]To investigate the effect of recombinant protein PPE25 of Mycobacterium avium on immune response of human THP-1 macrophages and the molecular mechanism related. [Method]Gene amplification,vector construction,induced expression and separation and purification were used to prepare recombinant PPE25 protein of Mycobacterium avium. After treated the THP-1 macrophages with the recombinant PPE25 protein,flow cytometry was used to detect the apoptosis rate of macrophage. ELISA was utilized to detect the level of TNF-α in culture supernatant. Western Blotting was conduceted to measure the protein expression of Bax,Bcl-2,TLR2 and TLR4. The mRNA expression of IL-6,IL-12 were conduceted by q PCR.[Result]Recombinant PPE25 protein was successfully prepared by E. coli expression system. After treated with the recombinant PPE25 protein,the apoptosis rate of macrophages was increased( P < 0. 05),the expression of Bax proteins was increased( P <0. 05),and the expression of Bcl-2 protein was decreased( P < 0. 05). In addition,the expressions of TLR2 and TLR4 were increased( P < 0. 05),the level of TNF-α was increased( P < 0. 05),and the expression of IL-6 and IL-12 were increased( P < 0. 05).[Conclusion]The Mycobacterium avium PPE25 protein could enhance the apoptosis of human THP-1 macrophages,and promote the secretion of inflammatory cytokines IL-6 and IL-12 by activating the TLR2 or TLR4 signaling pathway in macrophages,so as to induce the body to produce immune response and resist Mycobacterium avium infection.
作者
宁雪萍
田爽
龙倩
何会姣
陈蕴玟
凌敏
NING Xue-ping;TIAN Shuang;LONG Qian;HE Hui-jiao;CHEN Yun-wen;LING Min(Key Laboratory of Longevity and Aging-related Diseases,Ministry of Education,China,Laboratory of Biological Molecular Medicine Research of Guangxi University,Department of Biotechnology,Guangxi Medical University,Nanning 530021,China)
出处
《生物技术》
CAS
2021年第5期425-430,477,共7页
Biotechnology
基金
广西自然科学基金项目(“鸟分枝杆菌PPE25调控巨噬细胞免疫应答的机制研究”,2018GXNSFAA281023)。
