摘要
目的将肝素结合血凝素(HBHA)重组表达于耻垢分枝杆菌(MS),鉴定其对A549细胞自噬作用的影响。方法将HBHA克隆于穿梭表达质粒pMV261,构建HBHA重组载体,电转化至MS;感染A549细胞,Western blot法鉴定A549细胞微管相关蛋白轻链3(LC3)的表达情况,并检测LC3Ⅱ/LC3Ⅰ比值的变化可估计自噬的水平;利用单丹磺酰尸胺(MDC)将自噬体染色后,激光共聚焦观察细胞内自噬体变化的情况;通过MS、rMS-HBHA感染A549细胞18 h,计算胞内活菌数,鉴定感染状况。结果 rMS-HBHA经42℃热诱导成功表达HBHA蛋白;相对野生型MS,重组表达HBHA蛋白的MS(rMS-HBHA)可以显著抑制A549细胞中的LC3Ⅰ和LC3Ⅱ的表达,rMS-HBHA组的LC3Ⅱ/LC3Ⅰ比值为0.625显著低于MS组2.025,表明rMS-HBHA组自噬体形成受到抑制;并且rMS-HBHA感染A549细胞18 h后胞内活菌为6.3×106,明显高于MS组1×105。结论外源性HBHA可以通过抑制A549细胞的自噬作用增强MS的感染能力。
Objective To construct recombinant Mycobacterium smegmatis （MS） expressing heparin-binding hemagglutinin （HBHA） （rMS-HBHA） and identity its impact on autophagy in the A549 cells. Methods The HBHA was cloned into the shuttle expression plasmid pMV261. The HBHA recombinant vector was constructed and electrotransformed into MS. Western blotting was performed to detect the expression level of the microtubule-associated protein light chain 3 （ LC3 ） in A549 cells infected with rMS-HBHA, and the LC3-11/LC31 ratio was calculated to evaluate the level of autophagy. Then, autophagosomes were detected using monodansylcadaverine （MDC） staining and identified through laser confocal microscopy. Finally, the A549 cells that were infected with MS and rMS-HBHA for 18 hours were measured for the intracellular survival MS and rMS. Results HBHA protein was successfully expressed in rMS-HBHA under 42℃ heat-induction. Compared with wild-type MS, rMS-HBHA significantly inhibited the expressions of LC31 and LC311 in A549 cells. And LC311/LC31 ratio of the rMS-HBHA group （0. 625 ） was significantly lower than that of the MS group （ 2. 025 ）, indicating that autophagosome formation of the rMS-HBHA group was inhibited. In addition, the intracellular survival rMS-HBHA （6.3 ×10^6） was significantly higher than that of the MS group （ 1 ×10^5 ） in the infected A549 cells after 18 hours. Conclusion The exogenous HBHA could enhance the infection ability of MS in A549 cells through inhibiting autophagy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第7期696-699,703,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30872358)
