中国医科大学学报

中国医科大学学报

中国医科大学学报

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随机转座子插入构建甲型副伤寒杆菌启动子文库

许泽仰1,2,洪愉3,冯梦蝶1,毛普加1,赵继华3,杨洪文3,宋武战3,王纪爱2,黄芬1,井申荣1,曾韦锟1,   

  1. 1. 昆明理工大学医学院,昆明 650500;2. 昆明学院医学院临床检验教研室,昆明 650214;3. 成都军区昆明总医院核医学科,昆明 650032
  • 收稿日期:2015-01-25 出版日期:2016-01-05 发布日期:2015-12-30
  • 通讯作者: 曾韦锟,E-mail:zengweikun@126.com
  • 作者简介:许泽仰(1990 -),男,硕士研究生.
  • 基金资助:

    国家自然科学基金(31160193);云南省教育厅科学研究基金重点项目(2015Z168);云南省应用基础研究面上项目(2010ZC055;2012FB135)

Construction of a Promoter Library of Salmonella paratyphi A by Inserting Transposon

XU Ze-yang1,2,HONG Yu3,FENG Meng-die1,MAO Pu-jia1,ZHAO Ji-hua3,YANG Hong-wen3,SONG Wu-zhan3,WANG Ji-ai2,HUANG Fen1,JING Shen-rong1,ZENG Wei-kun1,2   

  1. 1. Medical Faculty,Kunming University of Science and Technology,Kunming 650500,China;2. Department of Clinical Laboratory,Medical Faculty,Kunming University,Kunming650214,China;3.Department of Nuclear Medicine,Kunming General Hospital of Chengdu Military Command,Kunming650032,China
  • Received:2015-01-25 Online:2016-01-05 Published:2015-12-30

摘要:

目的 构建携带氯霉素乙酰转移酶(CAT)的转座子自杀性质粒,通过接合转座获得甲型副伤寒沙门氏菌(副甲)启动子 文库。方法 PCR扩增CAT的基因亚克隆至pMD-18T载体,鉴定正确后酶切插入转座子自杀性质粒pSC189,获得新的转座子 载体pSC-CAT,将携带pSC-CAT的供体菌与副甲受体菌进行接合转座,涂布氯霉素平板筛选转座的细菌,以随机筛选出来的细 菌基因组为模板,热不对称PCR扩增插入位点侧翼序列,并进行测序、比对分析。结果 筛选获得约1000个突变菌,随机挑取 6个菌,转座子插入位点的上游分别对应6个可疑启动子区域。结论 通过自杀性转座子载体pSC-CAT可高效获得副甲菌启动 子文库,为进一步研究副甲菌基因表达与调控奠定了基础。

关键词: 甲型副伤寒沙门氏菌, 氯霉素乙酰转移酶, pSC189, 接合转移转座

Abstract:

Objective To establish a promoter library of Salmonella paratyphi A by transposition with new suicide plasmid with transposonwhich containing chloramphenicol acetyltransferaseCATas the report gene. Methods The CAT gene of was amplifed by PCR and cloned into pMD18-T vector. The above fragment was then inserted to suicide plasmid pSC189and the new plasmid was named pSC-CAT. After conjugation between S17-1λpir/pSC-CAT andSalmonella paratyphi Athe mixer was put on the plate containing chloramphenicol to screen the transductants. The genome of random picked bacteria was extracted as template of the thermal asymmetric interlaced PCRTail-PCRto amplify flanking sequence near insertion site. The PCR products were sequenced and analysed with blast. Results About 1 000 mutant strains were got. Six suspicious promoter regions were found in the front of inserted site from six random selected bacteria. Conclusion A promoter library of Salmonella paratyphi A could be effectively established through suicide vector pSC-CATwhich laid a foundation for further study of gene expression and regulation of Salmonella paratyphi A.

Key words: Salmonellaparatyphi A, chloramphenicol acetyltransferase, pSC189, conjugate transpose

中图分类号: 

  • R378.2
[1] 王齐晖, 刘学佳, 胡锦瑞, 褚云卓. 肠杆菌科细菌mcr-1基因筛查及其与多黏菌素耐药表型的相关性[J]. 中国医科大学学报, 2018, 47(8): 713-716.
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