细菌染色体第1类整合子捕获耐药性基因盒反应模型的构建

Construction of reaction model for class 1 integron capturing antibiotic resistance gene cassettes on chromosome of bacteria

【背景】整合子在细菌耐药性的获得及传播中占据重要地位,对于整合反应检测方法的改良及反应机制的研究,可以加深我们对细菌耐药性产生和播散的理解,为遏制耐药菌株的产生和播散提供新的途径。【目的】在细菌染色体上构建第1类整合子反应模型,用于评价整合酶介导的基因盒位点特异性重组。【方法】采用PCR分别扩增含氯霉素耐药基因cat的CM片段、含基因盒aadA5的LacA5片段、含整合子重组位点attI1及强可变区启动子的PcS片段和插入位点两侧的同源臂,重叠延伸聚合酶链反应连接上述5个片段制备整合子模型插入片段,通过同源重组将构建好的整合子模型片段插入大肠埃希菌JM109染色体中。转入高表达第1类整合酶的质粒pHSint,在链霉素平板上筛选发生整合的菌株,并用聚合酶链反应和测序验证。【结果】构建的整合子模型片段经测序与预期一致,整合子模型片段成功插入大肠埃希菌JM109染色体中。转入高表达整合酶的质粒pHSint后,在链霉素平板上成功筛选出基因盒aadA5发生整合的菌株,经聚合酶链反应扩增并测序与预期一致。【结论】在大肠埃希菌染色体上成功构建第1类整合酶介导基因盒位点特异性重组反应模型,为进一步揭示整合子捕获耐药性基因盒的反应机制奠定基础。

[Background] Integron plays important roles in acquisition and spread of antibiotic resistance among bacteria. The research on the improvement of detection methods and reaction mechanisms of integron integration reaction can deepen the understanding of the integron’s contribution in antibiotic resistance acquisition and spread, and provide new ways in suppression of the emergence and spread of the resistant strains. [Objective] To construct class 1 integron reaction model on bacterial chromosome to evaluate integrase-mediated gene cassette site-specific recombination. [Methods] The CM fragment containing chloramphenicol resistance gene cat, the LacA5 fragment containing gene cassette aadA5, the PcS fragment containing integron recombination site attI1 and strong promoter of variable region, the homologous arms on both sides of the knock-in site were amplified by polymerase chain reaction separately. The above five fragments were linked by overlap extension polymerase chain reaction to prepare the knock-in fragment of integron reaction model, and the constructed fragment was knocked into the chromosome of Escherichia coli JM109 by homologous recombination. After transferred with the class 1 integron integrase high expression plasmid pHSint, the integrated strains were screened on streptomycin plate and identified by polymerase chain reaction and sequencing. [Results] The sequencing results of the constructed fragment of integron reaction model were identity with that of expected. The constructed fragment was successfully knocked into the chromosome of E. coli JM109. After transferred with the integrase high expression plasmid pHSint, the strains with gene cassette aadA5 integrated into attI1 were successfully screened on streptomycin plate. The results of polymerase chain reaction and sequencing were identity with that of expected. [Conclusion] Reaction model of class 1 integron integrase-mediated gene cassette site-specific recombination was successfully constructed on E. coli chromosome. It will lay the foundation for further revealing the reaction mechanism of integron capture antibiotic resistance gene cassettes.