中文摘要：

目的探讨膜融合相关蛋白TECPR1对羊种布鲁氏菌16M感染小鼠巨噬细胞RAW264.7（RAW264.7）引起的非典型自噬及布鲁氏菌胞内生存繁殖能力的影响。方法根据AIR核苷酸序列,分别设计2条特异性小干扰RNA（short interfering RNA,siRNA）分子,亚克隆到慢病毒pLentiLox3.7载体,并转染RAW264.7,通过QRT-PCR筛选干扰效果最优的质粒;用布鲁氏菌感染干扰AIR后的RAW264.7,采用QRT-PCR检测细胞自噬相关基因p62、LC3-Ⅰ、LC3-Ⅱ的mRNA表达量,采用Western blot检测细胞自噬相关基因p62、LC3-Ⅰ、LC3-Ⅱ蛋白表达量;同时检测16M在干扰AIR后RAW264.7中的生存繁殖能力。结果获得2条对AIR干扰效果达70%以上特异性的siRNA分子,自噬相关基因LC3-Ⅰ、LC3-Ⅱ、p62的mRNA相对表达量分别为66%、77%和63%,差异有统计学意义（F值分别为21.52,25.38,10.87,P〈0.05）;LC3-Ⅰ、LC3-Ⅱ、p62蛋白相对表达量分别为45.71%、34.29%和46.13%,差异有统计学意义（F值分别是753.92,332.38,768.15,P〈0.01）;24h后干扰AIR后的RAW264.7中布鲁氏菌数量为8.58×105CFU,对照组为6.02×106 CFU,差异有统计学意义（F值为13.45,P〈0.05）。结论 AIR基因沉默可促进布鲁氏菌诱导的非典型自噬进一步成熟,布鲁氏菌胞内繁殖力显著降低,这可能是AIR结构域在自噬小体和溶酶体融合过程中发挥重要作用,该研究进一步解释了布鲁氏菌能够逃避巨噬细胞自噬-溶酶体降解途径的分子机制。

英文摘要：

Objective To examine the effects of TECPR1 on atypical autophagy induced by brucellosis and intracellular survival of Brucella in RAW264.7murine macrophages. Methods In accordance with the nucleotide sequence of AIR in the GenBank database,two specific short interfering RNA（siRNA）molecules targeting AIR were designed and cloned into the lentiviral vector pLentiLox3.7.This vector was transfected into RAW264.7cells,and QRT-PCR was used to screen for the plasmid with the greatest interference.QRT-PCR was also used to detect levels of mRNA of the autophagyrelated genes p62,LC3-Ⅰ,and LC3-Ⅱ.Western blotting was used to detect expression of those proteins,and colonyforming unit analysis was used to determine intracellular survival of the 16 Mstrain in RAW264.7cells after interference with AIR. Results Two specific siRNA molecules causing a high level of interference with AIR（more than 70%）were obtained.The levels of mRNA of the autophagy-related genes LC3 I,LC3II,and p62 were 66%,77% and 63%,respectively,in RAW264.7cells following interference with AIR.These levels differed significantly from those in normal RAW264.7cells（F：21.52,25.38,and 10.87,respectively;P〈0.05）.The levels of LC3-I,LC3-Ⅱ,and p62 protein were 45.71%,34.29%and 46.13%,respectively,in RAW264.7cells following interference with AIR.These levels differed significantly from those in normal RAW264.7cells（F：753.92,332.38,and 768.15,respectively;P〈0.01）.Twenty-four h after interference with AIR,RAW264.7cells had significantly fewer colonies of the 16 Mstrain in comparison to the negative control（cells with AIR interference：8.58×105 CFU;negative control：6.02×106 CFU;Fvalue：13.45,P〈0.05）. Conclusion Silencing of the AIR gene promotes further atypical autophagy induced by Brucella.The intracellular survival of Brucella decreases markedly,which is probably due to the important role of the AIR structural do-main in the fusion of autophagosomes and lysosomes.In addition,this study has further elucidated the molecular mecha