کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2092129 1546002 2015 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A new method for simultaneous gene deletion and down-regulation in Brucella melitensis Rev.1
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
A new method for simultaneous gene deletion and down-regulation in Brucella melitensis Rev.1
چکیده انگلیسی

In this study, our aim was to integrate an antisense expression cassette in bacterial chromosome for providing a long-term expression down-regulation in a bid to develop a new approach for simultaneous deletion and down-regulation of target genes in bacterial system. Therefore, we were used this approach for simultaneous deletion of the perosamine synthetase (per) gene and down-regulation of the virB1 expression in Brucella melitensis Rev.1. The per gene, which is one of the LPS O-chain coding genes, was replaced by homologous recombination with an antisense virB1 expression cassette together with kanamycin resistance cassette (kanR). Deletion of the per gene was characterized by PCR analysis and DNA sequencing. The expression of antisense virB1 cassette was confirmed by RT-PCR. Down-regulation of the virB1 mRNA expression was quantified by real-time RT-PCR using virB1 specific primers relative to the groEL reference gene. The survival rate of mutant strain was evaluated by CFU count in the BALB/c mice. The virB1 mRNA expression was down-regulated on average 10-fold in mutant strain as compared to parental strain. The loss of per gene function and decrease of the virB1 mRNA expression resulted in reduced entry and survival of the mutant Rev.1 strain in BALB/c mice splenocytes. We propose that this method can be used for simultaneous regulation of multiple genes expression.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Microbiological Research - Volume 170, January 2015, Pages 114–123
نویسندگان
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