pEASY-E1-Int1表达载体的点突变改造与整合酶的表达分析
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摘要
整合子—基因盒系统(Integron-gene cassette system)能使耐药基因在细菌种内和种间快速传播。在整合子—基因盒系统中起关键作用的是整合酶,本研究的目的在于克隆与NCBI公布的整合酶DNA序列完全一致的序列,并进一步表达纯化整合酶。采用PCR技术点突变改造pEASY-E1-Int1表达载体上的目的基因整合酶基因,共含5个突变位点,测序比对后,再连接与pEASY-E1 expression表达载体上,转化到BL21(DE3)感受态细胞中,不同浓度IPTG诱导优化表达,经聚丙烯酰氨凝胶电泳(SDS-PAGE)和Western blotting检测分析,结果得到表达产物分子量为33 kD与Ⅰ类整合子整合酶分子量一致的整合酶。本研究为下一步比较野生型和耐药性大肠杆菌整合酶活性提供基础指导。
Drug resistant genes were spread rapidly between bacteria and bacteria by the integron-gene cassette system. The integrase plays a key role in the integrin gene cassette system. The purpose of this study was to clone integrase DNA sequences that released identical DNA sequences by the NCBI. Further, it was expressed and purificated. 5 mutations site of the integrase gene were changed on the pEASY-E1-Int1 expression vector by the PCR point mutations technology. The sequence was identical with that published by NCBI. Then, it was ligated with the expression vector pEASY-E1, and was transformed into the competent cells Escherichia coli BL21 DE3.The recombinant plasmid was induced by different concentrations of IPTG. SDS-PAGE and Western Blotting were showed the molecular weight of the integrase was consistent with the class Ⅰ integrase 33 kD. The study provides basic guidance for the next step to compare the activity of integrase between wild and resistant Escherichia coli.
Drug resistant genes were spread rapidly between bacteria and bacteria by the integron-gene cassette system. The integrase plays a key role in the integrin gene cassette system. The purpose of this study was to clone integrase DNA sequences that released identical DNA sequences by the NCBI. Further, it was expressed and purificated. 5 mutations site of the integrase gene were changed on the pEASY-E1-Int1 expression vector by the PCR point mutations technology. The sequence was identical with that published by NCBI. Then, it was ligated with the expression vector pEASY-E1, and was transformed into the competent cells Escherichia coli BL21 DE3.The recombinant plasmid was induced by different concentrations of IPTG. SDS-PAGE and Western Blotting were showed the molecular weight of the integrase was consistent with the class Ⅰ integrase 33 kD. The study provides basic guidance for the next step to compare the activity of integrase between wild and resistant Escherichia coli.
引文
Corre ia M.,Boavida F.,Grosso F.,Salgado M.J.,Lito L.M.,Cristino J.M.,Mendo S.,and Duarte A.,2003,Molecular characterization of a new class 3 integron in Klebsiella pneumoniae,Antimicrob.Agents Chemother.,47(9):2838-2843
Hall R.M.,and Collis C.M.,1995,Mobile gene cassettes and integrons:capture and spread of genes by site specific recombination,Mol.Microbiol.,15(4):593-600
Huang Z.M.,Mi Z.H.,Qin L.,Chen Y.,Wu J.,Wu L.,and Shan H.,2005,Analysis of resistance and genotyping of aminoglycoside modifying enzymes in Enterobacter cloacae,Zhonghua Weishengwuxue He Mianyixue Zazhi(Chinese Journal Microbiology and Immunology),25(1):428-431(黄支密,糜祖煌,秦玲,陈榆,吴晶,仵蕾,单浩,20株阴沟肠杆菌耐药性及氨基糖苷类修饰酶基因分析,中华微生物学和免疫学杂志,25(1):428-431)
Jia F.,Liang Y.X.,and Yang J.L.,2015,Detection and analysis of the prevalence of integronsin clinical drug resistant Staphylococcus epidermidis,Zhongguo Weisheng Jianyan Zazhi(Chinese Journal of Health Laboratory Technology),25(19):3312-3314(贾芳,梁艳霞,杨江流,2015,临床耐药性表皮葡萄球菌整合子分子检测分析,中国卫生检验杂志,25(19):3312-3314)
Krin E.,Cambray G.,and Mazel D.,2014,The superintegron integrase and the cassette promoters are coregulated in Vibrio cholera,PLoS One,3(9):91194
Li P.H.,Guo X.R.,Peng J.Y.,Chen H.,Tang Z.Z.,Jin W.Q.,Wang H.,and Han X.Y.,2016,Improvingβ-glucosidase activity by site-directed mutagenesis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(8):2083-2091(李沛华,郭雪睿,彭继燕,陈惠,唐自钟,金伟琼,王欢,韩学易,2016,定点突变技术提高β-葡萄糖苷酶活性,基因组学与应用生物学,35(8):2083-2091)
Li L.M.,Yuan X.Y.,Wang M.Y.,Wang H.J.,Li Q.,and Zhu Y.M.,2014,The integron-gene cassette system and the mechanism of bacterial drug resistance:research progress,Zhongguo Weishengtaixue Zazhi(Chinese Journal of Microecology),26(2):246-248(李鲁明,袁晓燕,王明义,王红军,李芹,朱亚梅,整合子-基因盒系统与细菌耐药机制的研究进展,中国微生态学杂志,26(2):246-248)
Mazel D.,2006,Integrons:agents of bacterial evolution,Nat.Rev.Microbiol.,4(8):608-620
Rowe-Maqnus D.A.,and Mazel D.,2002,The role of integrons in antibiotic resistance gene capture,Int.J.Med.Microbiol.,292(2):115-125
Stokes H.W.,and Hall R.M.,1989,A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons,Mol.Microbiol.,3(12):1669-1683
Stokes H.W.,O'Gorman D.B.,Recchia G.D.,Parsekhian M.,and Hall R.M.,1997,Structure and function of 59-base element recombination sites associated with mobile gene cassettes,Mol.Microbiol.,26(4):731-745
Hall R.M.,and Collis C.M.,1995,Mobile gene cassettes and integrons:capture and spread of genes by site specific recombination,Mol.Microbiol.,15(4):593-600
Huang Z.M.,Mi Z.H.,Qin L.,Chen Y.,Wu J.,Wu L.,and Shan H.,2005,Analysis of resistance and genotyping of aminoglycoside modifying enzymes in Enterobacter cloacae,Zhonghua Weishengwuxue He Mianyixue Zazhi(Chinese Journal Microbiology and Immunology),25(1):428-431(黄支密,糜祖煌,秦玲,陈榆,吴晶,仵蕾,单浩,20株阴沟肠杆菌耐药性及氨基糖苷类修饰酶基因分析,中华微生物学和免疫学杂志,25(1):428-431)
Jia F.,Liang Y.X.,and Yang J.L.,2015,Detection and analysis of the prevalence of integronsin clinical drug resistant Staphylococcus epidermidis,Zhongguo Weisheng Jianyan Zazhi(Chinese Journal of Health Laboratory Technology),25(19):3312-3314(贾芳,梁艳霞,杨江流,2015,临床耐药性表皮葡萄球菌整合子分子检测分析,中国卫生检验杂志,25(19):3312-3314)
Krin E.,Cambray G.,and Mazel D.,2014,The superintegron integrase and the cassette promoters are coregulated in Vibrio cholera,PLoS One,3(9):91194
Li P.H.,Guo X.R.,Peng J.Y.,Chen H.,Tang Z.Z.,Jin W.Q.,Wang H.,and Han X.Y.,2016,Improvingβ-glucosidase activity by site-directed mutagenesis,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(8):2083-2091(李沛华,郭雪睿,彭继燕,陈惠,唐自钟,金伟琼,王欢,韩学易,2016,定点突变技术提高β-葡萄糖苷酶活性,基因组学与应用生物学,35(8):2083-2091)
Li L.M.,Yuan X.Y.,Wang M.Y.,Wang H.J.,Li Q.,and Zhu Y.M.,2014,The integron-gene cassette system and the mechanism of bacterial drug resistance:research progress,Zhongguo Weishengtaixue Zazhi(Chinese Journal of Microecology),26(2):246-248(李鲁明,袁晓燕,王明义,王红军,李芹,朱亚梅,整合子-基因盒系统与细菌耐药机制的研究进展,中国微生态学杂志,26(2):246-248)
Mazel D.,2006,Integrons:agents of bacterial evolution,Nat.Rev.Microbiol.,4(8):608-620
Rowe-Maqnus D.A.,and Mazel D.,2002,The role of integrons in antibiotic resistance gene capture,Int.J.Med.Microbiol.,292(2):115-125
Stokes H.W.,and Hall R.M.,1989,A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons,Mol.Microbiol.,3(12):1669-1683
Stokes H.W.,O'Gorman D.B.,Recchia G.D.,Parsekhian M.,and Hall R.M.,1997,Structure and function of 59-base element recombination sites associated with mobile gene cassettes,Mol.Microbiol.,26(4):731-745