【摘要】：目的探討膜融合相關(guān)蛋白TECPR1對(duì)羊種布魯氏菌16M感染小鼠巨噬細(xì)胞RAW264.7(RAW264.7)引起的非典型自噬及布魯氏菌胞內(nèi)生存繁殖能力的影響。方法根據(jù)AIR核苷酸序列,分別設(shè)計(jì)2條特異性小干擾RNA(short interfering RNA,siRNA)分子,亞克隆到慢病毒pLentiLox3.7載體,并轉(zhuǎn)染RAW264.7,通過(guò)QRT-PCR篩選干擾效果最優(yōu)的質(zhì)粒;用布魯氏菌感染干擾AIR后的RAW264.7,采用QRT-PCR檢測(cè)細(xì)胞自噬相關(guān)基因p62、LC3-Ⅰ、LC3-Ⅱ的mRNA表達(dá)量,采用Western blot檢測(cè)細(xì)胞自噬相關(guān)基因p62、LC3-Ⅰ、LC3-Ⅱ蛋白表達(dá)量;同時(shí)檢測(cè)16M在干擾AIR后RAW264.7中的生存繁殖能力。結(jié)果獲得2條對(duì)AIR干擾效果達(dá)70%以上特異性的siRNA分子,自噬相關(guān)基因LC3-Ⅰ、LC3-Ⅱ、p62的mRNA相對(duì)表達(dá)量分別為66%、77%和63%,差異有統(tǒng)計(jì)學(xué)意義(F值分別為21.52,25.38,10.87,P0.05);LC3-Ⅰ、LC3-Ⅱ、p62蛋白相對(duì)表達(dá)量分別為45.71%、34.29%和46.13%,差異有統(tǒng)計(jì)學(xué)意義(F值分別是753.92,332.38,768.15,P0.01);24h后干擾AIR后的RAW264.7中布魯氏菌數(shù)量為8.58×105CFU,對(duì)照組為6.02×106 CFU,差異有統(tǒng)計(jì)學(xué)意義(F值為13.45,P0.05)。結(jié)論 AIR基因沉默可促進(jìn)布魯氏菌誘導(dǎo)的非典型自噬進(jìn)一步成熟,布魯氏菌胞內(nèi)繁殖力顯著降低,這可能是AIR結(jié)構(gòu)域在自噬小體和溶酶體融合過(guò)程中發(fā)揮重要作用,該研究進(jìn)一步解釋了布魯氏菌能夠逃避巨噬細(xì)胞自噬-溶酶體降解途徑的分子機(jī)制。
[Abstract]:Objective to investigate the effects of membrane fusion associated protein (TECPR1) on atypical autophagy induced by murine macrophage RAW264.7 (RAW264.7) and the viability and reproduction of Brucella spp. Methods according to the nucleotide sequence of AIR, two specific small interfering RNA (short interfering siRNAs were designed and subcloned into lentivirus pLentiLox3.7 vector and transfected into RAW264.7. The best interference plasmid was screened by QRT-PCR. RAW264.7 was infected with Brucella. The expression of autophagy associated gene p62LC3- 鈪，

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