pEASY-E1-Int1表达载体的点突变改造与整合酶的表达分析

To Reform the p EASY-E1-Int1 Expression Vector in the Point Mutation and Analyze the Expression of the Integrase

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 classⅠintegrase 33 kD.The study provides basic guidance for the next step to compare the activity of integrase between wild and resistant Escherichia coli.