[1]刘 冉,陈福广,陈 平,等.利用信息肽诱导和Cre/LoxP系统构建猪链球菌基因缺失株[J].中国预防兽医学报,2019,(02):125-130.[doi:0.3969/j.issn.1008-0589.201810003]
 LIU Ran,CHEN Fu-guang,CHEN Ping,et al.Gene deletion of Streptococcus suis by combination of pheromone induction and Cre/LoxP system[J].Chinese journal of preventive veterinary medicine,2019,(02):125-130.[doi:0.3969/j.issn.1008-0589.201810003]
点击复制

利用信息肽诱导和Cre/LoxP系统构建猪链球菌基因缺失株()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年02
页码:
125-130
栏目:
诊断技术
出版日期:
2019-02-15

文章信息/Info

Title:
Gene deletion of Streptococcus suis by combination of pheromone induction and Cre/LoxP system
文章编号:
1008-0589(2019)02-0125-06
作者:
 

刘 冉12陈福广2陈 平2黄萌萌2朱金鲁2谢 芳2孟庆文2刘思国2刘建华1*张跃灵2*

 (1. 新疆农业大学 动物医学院,新疆 乌鲁木齐 8300522;
2. 中国农业科学院哈尔滨兽医研究所 兽医生物技术国家重点实验室,黑龙江 哈尔滨 150069)
Author(s):
 

LIU Ran12 CHEN Fu-guang2 CHEN Ping2 HUANG Meng-meng2 ZHU Jin-lu2 XIE Fang2MENG Qing-wen3 LIU Si-guo2 LIU Jian-hua1* ZHANG Yue-ling2*

 (1. College of Veterinary Medicine, Xinjiang Agricultural University Wulumuqi 8300533, China; 2. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China)
关键词:
猪链球菌信息肽诱导Cre/LoxP系统基因缺失
Keywords:
 Streptococcus suis  pheromone induction  Cre/LoxP system  gene deletion
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201810003
文献标志码:
A
摘要:
为建立一种高效的猪链球菌(Streptococcus suis)基因缺失方法,本研究利用信息肽诱导DNA片段转化和Cre/LoxP系统去除抗性基因这两种技术,通过在S.suis 05ZYH33的ssu05_1921基因上、下游片段和壮观霉素抗性基因(spc)片段之间引入LoxP位点,采用信息肽GE9诱导构建的DNA片段转化S.suis并发生重组,经抗性筛选快速获得spc替代ssu05_1921基因的缺失株;进一步引入pSET6s/PtufA-cre质粒表达Cre重组酶作用于LoxP位点,去除spc基因,产生无痕ssu05_1921基因缺失株,经测序和RT-PCR验证缺失正确。本研究建立的该方法简单、快捷、阳性率高,为构建S.suis基因缺失株、研究S.suis致病机制提供了新的选择。
Abstract:
In the present study, an efficient method for gene-deletion of Streptococcus suis was established by combination of recently reported pheromone-induced DNA fragment transformation and Cre/LoxP system. Briefly, a DNA fragment was prepared by fusion of the upstream fragment of ssu05_1921 gene of S.suis, the spectinomycin resistance (spc) marker gene and the downstream fragment of ssu05_1921 gene. During the fusion processing, LoxP sites were introduced sequentially at 5’ and 3’ ends of the spc gene. Next, the DNA fragment was transformed into S.suis 05ZYH33 strain induced by the pheromone for recombin- ation, resulting in ssu05_1921-deleted from the S.suis strain in which the ssu05_1921 gene was substituted by spc marker gene. Then,  the spc marker gene flanked with LoxP sites was removed, leading to a marker-less ssu05_1921-deleted strain by introducing the Cre recombinase expressing recombinant plasmid of pSET6s/PtufA-cre. It was further verified that the ssu05_1921 gene was deleted by sequencing and RT-PCR. The results showed that this method is simple and rapid, with high positive rate. It provides a efficient alternative protocol for gene-deleted strain construction of S.suis,  facilitatinge further pathogenesis study of the pathogen.

参考文献/References:

[1]Staats J J, Feder I, Okwumabua O, et al. Streptococcus suis: Past and present [J]. Vet Res Commun, 1997, 21(6): 381-407.
[2]Lun Zhao-Rong, Wang Qiao-Ping, Chen Xiao-Guang, et al. Streptococcus suis: an emerging zoonotic pathogen [J]. Lancet Infect Dis, 2007, 7(3): 201-209.
[3]Huong V T L, Ha N, Huy N T, et al. Epidemiology, clinical manifestations, and outcomes of Streptococcus suis infection in humans [J]. Emerg Infect Dis, 2014, 20(7): 1105-1114.
[4]陆承平,姚火春,范红结,等. 猪链球菌致病性研究及其公 共卫生意义[J]. 中国预防医学杂志,2006,(04):361-362.
[5]Shanker E, Morrison D A, Talagas A, et al. Pheromone recognition and selectivity by ComR proteins among Streptococcus species [J]. PLoS Pathog, 2016, 12(12): e1005979.
[6]Claverys J P, Dintilhac A, Mortier-Barriere I, et al. Regulation of competence for genetic transformation in Streptococcus pneumoniae [J]. J Appl Microbiol, 1997, 83(S1): 32S-41S.
[7]Pestova E V, Havarstein L S, Morrison D A. Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system [J]. Mol Microbiol, 1996, 21(4): 853-862.
[8]Mashburn-Warren L, Morrison D A, Federle M J. The cryptic competence pathway in Streptococcus pyogenes is controlled by a peptide pheromone [J]. J Bacteriol, 2012, 194(17): 4589- 4600.
[9]Ahn S J, Wen Z T, Burne R A. Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159 [J]. Infect Immun, 2006, 74(3): 1631-1642.
[10]Kowalko J E, Sebert M E. The Streptococcus pneumoniae competence regulatory system influences respiratory tract colonization [J]. Infect Immun, 2008, 76(7): 3131-3140.
[11]Zaccaria E, van Baarlen P, de Greeff A, et al. Control of competence for DNA transformation in Streptococcus suis by genetically transferable pherotypes [J]. PLoS One, 2014, 9(6).
[12]Sternberg N, Hamilton D, Hoess R. Bacteriophage P1 site-specific recombination. II. Recombination between loxP and the bacterial chromosome [J]. J Mol Biol, 1981, 150(4): 487-507.
[13]Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination. I.Recombination between loxP sites [J]. J Mol Biol, 1981, 150(4): 467-486.
[14]Albert H, Dale E C, Lee E, et al. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome [J]. Plant J, 1995, 7(4): 649-659.
[15]Koczula A, Willenborg J, Bertram R, et al. Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis [J]. J Microbiol Meth, 2014, 10780-83.
[16]Chen Chen, Tang Jia-Qi, Dong Wei, et al. A glimpse of Streptococcal toxic shock syndrome from comparative genomics of S-suis 2 Chinese isolates [J]. PLoS One, 2007, 2(3).
[17]Takamatsu D, Osaki M, Sekizaki T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis [J]. Plasmid, 2001, 46(2): 140-148.
[18]Rocha D J, Santos C S, Pacheco L G. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis [J]. Anton Leeuw, 2015, 108(3): 685-693.
[19]任建鸾,陆承平. 猪链球菌9型GZ0565株体外传代的生物学稳定性[J]. 中国兽医科学,2010,40(11):1115-1118.
[20]Robb M, Hobbs J K, Woodiga S A, et al. Molecular characterization of N-glycan degradation and transport in Streptococcus pneumoniae and its contribution to virulence [J]. PLoS Pathog, 2017, 13(1): e1006090.
                                                 (本文编辑:彭永刚)

相似文献/References:

[1]苑青艳,王 强,李晓云,等.猪源链球菌对环丙沙星耐药性与耐药基因突变的相关性研究[J].中国预防兽医学报,2009,(03):201.
 YUAN Qing-yan,WANG Qiang,LI Xiao-yun,et al.Relationship between ciprofloxacin resistance and gyrA, parC gene mutation in Streptococcus suis [J].Chinese journal of preventive veterinary medicine,2009,(02):201.
[2]李春玲,余炜烈,王贵平,等.猪链球菌扁桃体分离株的毒力因子分布特征与致病性[J].中国预防兽医学报,2009,(02):104.
 LI Chun-ling,YU Wei-lie,WANG Gui-ping,et al.Genetype and pathogenicity of Streptococcus suis isolated from palatine tonsils of slaughter pigs in China[J].Chinese journal of preventive veterinary medicine,2009,(02):104.
[3]徐 敏,王淑杰,蔡雪辉*,等.猪链球菌重组GDH蛋白间接ELISA检测方法的建立 [J].中国预防兽医学报,2009,(08):623.
 XU Min,WANG Shu-jie,CAI Xue-hui*,et al.Development of an indirect-ELISA for detection of antibodies against Streptococcus suis using recombinant GDH protein as antigen [J].Chinese journal of preventive veterinary medicine,2009,(02):623.
[4]刘 洋,徐 刚,王 敏,等.酒石酸泰乐菌素联合阿莫西林钠缩小猪链球菌耐药突变选择窗的研究[J].中国预防兽医学报,2010,(01):65.
 LIU Yang,XU Gang,WANG Min,et al.Narrowing mutant selection window of Streptococcus suis by combination use of tylosin tartrate and amoxicillin sodium in vitro[J].Chinese journal of preventive veterinary medicine,2010,(02):65.
[5]李晓云,赵志慧,闫 明,等.东北地区猪链球菌对大环内酯类药物耐药机制的研究[J].中国预防兽医学报,2008,(08):612.
 LI Xiao-yun,ZHAO Zhi-hui,YAN Ming,et al.Mechanism of Streptococcus suis resistance to Macrolides in Northeast China[J].Chinese journal of preventive veterinary medicine,2008,(02):612.
[6]李春玲,余炜烈,贾爱卿,等.应用多重PCR检测屠宰猪扁桃体中的猪链球菌[J].中国预防兽医学报,2008,(05):344.
 LI Chun-ling,YU Wei-lie,JIA Ai-qing,et al.Carrier rate of Streptococcus suis in palatine tonsils of slaughter pigs in south China[J].Chinese journal of preventive veterinary medicine,2008,(02):344.
[7]高志强,宋 立,张鹤晓,等.猪链球菌通用及2型TaqMan荧光PCR检测技术建立与应用[J].中国预防兽医学报,2007,(07):545.
 GAO Zhi-qiang,SONG Li,ZHANG He-xiao,et al.Development and application of TaqMan real-time PCR for Streptococcus suis and S.suis type 2[J].Chinese journal of preventive veterinary medicine,2007,(02):545.
[8]王淑杰,徐 敏,刘永刚,等.一株血清7型致病性猪链球菌的分离鉴定[J].中国预防兽医学报,2011,(10):763.[doi:10.3969/j.issn.1008-0589.2011.10.03]
 WANG Shu-jie,XU Min,LIU Yong-gang,et al.Isolation and identification of one pathogenic Streptococcus Suis 7 serotype[J].Chinese journal of preventive veterinary medicine,2011,(02):763.[doi:10.3969/j.issn.1008-0589.2011.10.03]
[9]金家民,王淑杰,高明明,等.猪链球菌7型强弱毒株SBP_bac_5差异表达蛋白的鉴定[J].中国预防兽医学报,2013,(03):0.
 JIN Jia-min,WANG Shu-jie,GAO Ming-ming,et al.Identification of the differential expression of bacterial extracellular solute-binding protein family 5 in Streptococcus suis 7 virulent and attenuated strains[J].Chinese journal of preventive veterinary medicine,2013,(02):0.
[10]高明明,王淑杰,金家民,等.一株致病性血清2型猪链球菌的多位点序列分型及其灭活后对小鼠的免疫保护效果评价[J].中国预防兽医学报,2015,(01):57.[doi:10.3969/j.issn.1008-0589.2015.01.14]
 GAO Ming-ming,WANG Shu-jie,JIN Jia-min,et al.MLST genotyping analysis of a pathogenic Streptococcus suis and evaluation of the immune protection in mice[J].Chinese journal of preventive veterinary medicine,2015,(02):57.[doi:10.3969/j.issn.1008-0589.2015.01.14]

更新日期/Last Update: 2019-03-06