Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach
Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach
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DOI：
10.1016/j.micres.2013.12.008
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发表时间：
2014-01-01
影响因子：
6.7
通讯作者：
Bose, Mridula
中科院分区：
文献类型：
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作者：
Chandolia, Amita;Rathor, Nisha;Bose, Mridula
反义策略是基因沉默所产生的基因敲除突变的有吸引力的替代品。MCE基因已被证明与分枝杆菌的摄取和细胞内的生存有关。在这里，我们报告了利用反义技术降低结核分枝杆菌mce4A和mce1A基因的表达。为此，将mce4A和mce1A的1.1kb片段反向克隆到pSD5穿梭载体中，分别构建了反义载体pSD5-4AS和pSD5-1AS。在结核分枝杆菌H37Rv中，Mce4A的表达减少了约60%，Mce1A蛋白的表达减少了66%。我们还观察到，与只含有pSD5的结核分枝杆菌相比，这两种反义菌株的细胞内生存能力都显著降低。RT-PCR分析表明，反义基因不改变各自操纵子MCEA基因上游和下游的转录。反义构建物的菌落形态、体外生长特性和药物敏感性没有变化。这些结果表明，反义技术是一种很有前途的多单位操纵子基因功能分配方法，可以作为一种合适的策略应用。(C)2014年爱思唯尔股份有限公司。版权所有。
Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy. (C) 2014 Elsevier GmbH. All rights reserved.