|
|
|
| Epitope Tagging of the hfq Gene in Brucella melitensis by Suicide Plasmid |
| CUI Ming-quan1,2, XU Jie2,3, ZHANG Ting-ting1, WANG Tong-kun2, SHANG Wei2, CHEN Ze-liang2, YUAN Jing2, DU Xin-ying2, WANG Zhou-jia2, KE Yue-hua2, ZHONG Zhi-jun1, YUAN Xi-tong2, HUANG Liu-yu2, PENG Guang-neng1, WANG Yu-fei2 |
1. College of Veterinary Medicine, Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya'an 625014, China;
2. Institute of Disease Control and Prevention, Academy of Military Medical Science, Beijing 100071, China;
3. School of Public Health, Jilin University, Changchun 130021, China |
|
|
|
Abstract Epitope tagging of chromosomal gene hfq in Brucella melitensis were constructed by suicide vector to study the function of the RNA molecular chaperone Hfq. The epitope-tagging cassettes were obtained by PCR amplification from the genomic DNA of B. melitensis 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 pMD18-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.
|
|
Received: 01 August 2012
Published: 25 November 2012
|
|
|
|
Cite this article:
CUI Ming-quan, XU Jie, ZHANG Ting-ting, WANG Tong-kun, SHANG Wei, CHEN Ze-liang, YUAN Jing, DU Xin-ying, WANG Zhou-jia, KE Yue-hua, ZHONG Zhi-jun, YUAN Xi-tong, HUANG Liu-yu, PENG Guang-neng, WANG Yu-fei. Epitope Tagging of the hfq Gene in Brucella melitensis by Suicide Plasmid. China Biotechnology, 2012, 32(11): 75-80.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2012/V32/I11/75
|
|
|
[1] Boschiroli M L, Foulongne V, O'Callaghan D. Brucellosis: a worldwide zoonosis. Curr Opin Microbiol, 2001, 4(1): 58-64.
[2] Celli J. Survivng inside a macrophage: The many ways of Brucellae. Res Microbiol, 2006, 157(2): 93-98.
[3] Sun X, Wartell R M. Escherichia coli Hfq binds A18 and DsrA domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites. Biochemistry, 2006, 45(15): 4875-4887.
[4] Valentin-Hansen P, Eriksen M, Udesen C, et al. The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol, 2004, 51(6): 1525-1533.
[5] Sittka A, Pfeiffer V, Tedin K, et al. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol Microbiol, 2007, 63(1): 193-217.
[6] Tsui H C, Leung H C, Winkler M E. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol, 1994, 13(1): 35-49.
[7] Sonnleitner E, Schuster M, Sorger-Domenigg T, et al. Hfq-dependent alterations of the transcriptome profile and effects on quorum sensing in Pseudomonas aeruginosa. Mol Microbiol, 2006, 59(5): 1542-1558.
[8] 王玉飞, 陈泽良, 赵红庆, 等. 以克隆载体为自杀载体快速构建布鲁氏菌的无痕缺失突变株. 微生物学通报, 2007, 34(4): 642-645. Wang Y F, Chen Z L, Zhao H Q, et al. Construction of brucella unmarked deletion mutant by using conventional cloning vector as suicide plasmid. Microbiology, 2007, 34(4): 642-645.
[9] Giaever G, Chu A M, Ni L, et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature, 2002, 418(6896): 387-391.
[10] 白耀霞, 杨毅, 王玉飞,等. 利用T载体克隆快速构建布鲁氏菌缺失突变株. 中国生物工程杂志, 2010, 30(9): 62-67. Bai Y X, Yang Y, Wang Y F, et al. Construction of Brucella deletion mutant by using T cloning vector. China Biotechnology, 2010, 30(9): 62-67. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
