利用自杀载体构建布鲁氏菌hfq表位标记菌株  

Epitope Tagging of the hfq Gene in Brucella melitensis by Suicide Plasmid

作　　者：崔明全[1,2] 徐杰[2,3] 张婷婷[1] 王同坤[2] 尚伟[2] 陈泽良[2] 袁静[2] 杜昕颖[2] 汪舟佳[2] 柯跃华[2] 钟志军[1] 苑锡铜[2] 黄留玉[2] 彭广能[1] 王玉飞[2] 

机构地区：[1]四川农业大学动物医学院动物疫病与人类健康四川省重点实验室,雅安625014 [2]军事医学科学院疾病预防控制所,北京100071 [3]吉林大学公共卫生学院,长春130021

出　　处：《中国生物工程杂志》2012年第11期75-80,共6页China Biotechnology

基　　金：国家自然科学基金(81171530;31000041);教育部"长江学者和创新团队发展规划"创新团队项目(IRT0848)资助项目

摘　　要：目的:建立一种利用自杀载体构建布鲁氏菌hfq表位标记菌株的方法。方法:在hfq的反向引物带上FLAG或HIS标签序列,以布鲁氏菌Brucella melitensis 16M为模板扩增出3'端带有标签的hfq标记盒。将其直接与布鲁氏菌的自杀载体pMD18-T vector连接,获得带有标签的标记载体,将载体转入布鲁氏菌感受态细胞并筛选抗性克隆,进而获得表位标签与目的基因C末端融合的重组子。利用RT-PCR和Western blot分析C末端融合了表位标签的目的蛋白的转录和表达的情况。结果:获得了布鲁氏菌的表位标记菌株16M-T-HfqFLAG和16M-T-HfqHIS。RT-PCR实验结果表明带有标签的hfq基因可以转录,Western blot实验结果证实利用针对FLAG或HIS标签的抗体能够检测到Hfq蛋白的表达。结论:利用自杀载体可以快速构建布鲁氏菌的表位标记菌株,这不仅为Hfq的功能研究奠定了基础,也为布鲁氏菌的基因功能研究提供了一个有价值的研究手段。Epitope tagging of chromosomal gene hfq in Brucella melitensis were constructed by suicide vector to study the function of the RNA molecular chaperone I-Ifq. The epitope-tagging cassettes were obtained by PCR amplification from the genomic DNA of B. melltensis 16M with primers that carry homologous to the targeted gene hfq and FLAG or HIS encoding sequences （reverse primer）. Then, the epitope-tagging cassettes were ligated with pMDI$-T vector, resulted in epitope-tagging plasmids. The tagging plasmids were transformed into recombination competent cells and C-terminal-tagged recombinants were selected by ampicillin resistance. The transcription of resulting C-terminal-tagged hfq was identified by RT-PCR. And the expression of the tagged Hfq was detected indirectly by the antibodies against FLAG or HIS tag. As the results showed, it is a convenient method for adding an epitope-encoding tail to the interesting gene in the Brucella chromosome. The method described here provides a powerful tool in the functional study of Brucella genes.

关 键 词：布鲁氏菌 自杀载体 表位标记 HFQ 

分 类 号：Q813.1[生物学—生物工程]