接合转移质粒pUCP24T的构建与性能研究  

Construction and characterization of transfer plasmid pUCP24T

作　　者：陈树林[1] 陈茶[2] 黄彬[1] 张妮[2] 鄂顺梅[2] 蔡壬辛[1] 袁慧[2] 曲平华[2] 林冬玲[2] 

机构地区：[1]中山大学附属第一医院检验医学部,广东广州510080 [2]广东省中医院检验医学部,广东广州510006

出　　处：《中国微生态学杂志》2013年第7期771-774,共4页Chinese Journal of Microecology

基　　金：国家自然科学基金资助项目(81071397;81271909);广东省医学科研基金资助项目(A2011168);广东省科技计划项目(2011B031800037)

摘　　要：目的构建能在大肠埃希菌(E.coli)和铜绿假单胞菌(PA)间高效接合转移的质粒。方法通过NCBI blastn程序分析质粒pCVD442接合反应起始序列oriT,PCR扩增oriT后插入pMD18-T,再将pMD18-oriT转化E.coli DH5α,经酶切、测序验证后用SmaⅠ和HindIIII双酶切亚克隆至质粒pUCP24,获得质粒pUCP24T,将pUCP24T转化E.coli后研究pUCP24T从E.coli到PA的接合效率和质粒稳定性等。结果成功构建pUCP24T重组质粒,在E.coli和PA的接合实验中,E.coli(pUCP24T)-PA共培养2 h的接合效率平均为3.588×10-2,按12 h一代连续9代继代培养108 h,抗生素压力下质粒保存率为98.29%,无抗生素的培养基中质粒保存率为21.76%。结论成功构建高接合效率的接合转移质粒pUCP24T。Objective To construct a higher conjugative transfer plasmid pUCP24T that could transfer between Escherichia coli（E.coli） and Pseudomonas aeruginosa（PA）.Methods Plasmid pCVD442 oriT（origin of transfer） was analyzed by NCBI blastn program;the fragment oriT was amplified by PCR and inserted into pMD18-T vector to form pMD18-oriT.It was transformed into E.coli DH5α,and identified by restriction enzyme digestion and sequencing.Then pMD18-oriT was digested by SmaI and HindIIII and the oriT fragment was inserted into pUCP24 to form recombinant plasmid pUCP24T.The pUCP24T was transformed to E.coli SM10λpir to investigate the conjugation efficiencies of parental recipient strains and its stability in recipient PAO1 strain.Results The recombinant plasmid pUCP24T was successfully constructed.During the E.coli-PA parental mating experiments,the conjugation efficiency was 3.588×10-2 when E.coli（pUCP24T）-PA were co-cultured for 2 hours at 37℃.The plasmid stability assay was performed by 9 continuous passages at 12 hour interval,totally 108 hours.The rate of stable plasmid was 98.29% at selective pressure,while 21.76% at nonselective pressure.Conclusion The recombinant plasmid pUCP24T was constructed successfully with higher conjugation efficiency.

关 键 词：铜绿假单胞菌 接合性质粒 基因水平转移 

分 类 号：R378.991[医药卫生—病原生物学]