多重耐药维氏气单胞菌porin基因敲除的构建及其耐药特性研究

Construction of a porin knockout strain of multidrug-resistant Aeromonas veronii and its drug resistance characteristics

目的研究微孔蛋白基因porin对维氏气单胞菌(Aeromonas veronii,A.veronii)耐药性的影响以解析A.veronii的耐药机制。方法以多重耐药的A.veronii WL-3为研究对象,采用同源重组双交换的方法构建Δporin敲除株,利用比浊法测定其生长曲线,通过药敏纸片法分析其耐药性。结果以A.veronii WL-3基因组DNA为模板,利用overlap聚合酶链式反应技术扩增获得上下同源臂融合片段,并连接至自杀质粒pDM4中,重组成敲除质粒pDM4-Δporin。将pDM4-Δporin转化至感受态大肠杆菌SM10中,通过接合转移转入A.veronii WL-3中。通过同源重组双交换构建A.veronii WL-3Δporin敲除株。生长曲线显示与野生株相比,Δporin菌株的生长速度并无明显变化;抗生素耐药特征分析显示敲除porin基因会降低A.veronii对头孢氨苄、新霉素、四环素、环丙沙星等10种抗生素的敏感性,提高对多粘菌素B的敏感性。结论基因porin不是A.veronii生长所必需的,但会影响A.veronii对部分抗生素的敏感度。本研究所构建的敲除株可为深入研究porin在A.veronii的耐药机制提供必要材料。

Objective To study the effects of the porin gene on antibiotic resistance of Aeromonas veronii(A.veronii)and elucidate the mechanism of drug resistance of A.veronii.Methods TheΔporin knockout strain of multidrug-resistant A.veronii WL-3 was constructed by the homologous recombination double-exchange technology.The growth curves were measured by turbidimetric method and the antimicrobial susceptibility was analyzed by disk method.Results The upstream and downstream homologous fragments of the porin gene were amplified by polymerase chain reaction using the genomic DNA of A.veronii WL-3 as a template.A ligation fragment of both upstream and downstream of porin gene were obtained by overlap PCR amplification and ligated to the suicide plasmid pDM4 to construct the recombinant knockout plasmid pDM4-Δporin.pDM4-Δporin was transformed into Escherichia coli SM10 competent cell and then transferred to A.veronii WL-3 by conjugation.TheΔporin knockout strain of A.veronii WL-3 was formed by homologous recombinant double exchange.The growth curve showed that deletion of porin gene caused no significant growth retardation in A.veronii.Analysis of antibiotic resistance characteristics showed that knockout of the porin gene reduced the sensitivity of A.veronii to 10 kinds of antibiotics such as cephalexin,neomycin,tetracycline,ciprofloxacin and increased the sensitivity to polymyxin B.Conclusion The porin gene is not necessary for the growth of A.veronii,but affects the sensitivity of A.veronii to some antibiotics.The knockout strains constructed in this study can provide the necessary material for the function of porin in the antibiotic resistance mechanisms of A.veronii in the future.