Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA
Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA
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DOI：
10.1016/j.mimet.2004.01.010
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发表时间：
2004-05-01
影响因子：
2.2
通讯作者：
Tizard, MLV
中科院分区：
文献类型：
--
作者：
Granger, K;Moore, RJ;Tizard, MLV
RNA已被提取和分析，从体内衍生的鸟分枝杆菌亚种副结核杆菌从自然宿主中回收。从两只表现出约翰氏病临床症状的山羊的肠组织中选择性地提取细菌。快速取出小肠，冲洗掉管腔内容物，收获粘膜和粘膜下层。通过等渗裂解和差速离心从巨噬细胞中释放该材料中的分枝杆菌。提取RNA并与从体外生长的细菌中提取的RNA进行比较。采用实时荧光定量聚合酶链反应（Real-time polymerase chain reaction，RT-PCR）技术，从细菌的信使RNA中扩增出katG基因。编码推定的过氧化氢酶/过氧化物酶的katG RNA在体内和体外衍生的样品中显示出差异表达。我们假设体内来源的M.副结核病可能代表对巨噬细胞内环境中遇到的氧化应激的反应。(C)2004年由Elsevier B. V.出版
RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG RNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment. (C) 2004 Published by Elsevier B.V.