摘要
目的 利用同源重组技术构建肺炎克雷伯菌ampG基因缺失突变株,初步探讨ampG基因缺失对肺炎克雷伯菌AmpC酶诱导表达的影响. 方法 PCR分别扩增实验菌株ampG上下游同源臂片段,再利用重叠延伸PCR（gene splicing by overlap extension PCR ,SOE-PCR）技术获得ampG基因上下游同源臂融合片段,酶切后克隆至温敏性自杀载体pKO3-km上,构建pKO3-km-ΔampG重组质粒,电转化至肺炎克雷伯菌Kp1和Kp NTUH-K2044 菌株中,通过同源重组技术,利用pKO3-km的温敏特点,筛选出肺炎克雷伯菌ampG基因缺失的突变株;将克隆质粒pACYC184-ampCR分别导入Kp NTUH-K2044的野生型及其ampG基因缺失菌株中,使其获得AmpC酶基因;采用纸片扩散法,以头孢西丁为诱导剂,观察ampG基因缺失前后对肺炎克雷伯菌AmpC酶诱导表达的影响. 结果 成功构建pKO3-km-ΔampG重组质粒,经PCR及DNA测序证实获得肺炎克雷伯菌ampG基因缺失突变株,与野生型相比,Kp1菌株ampG基因敲除后AmpC酶诱导表型消失,而在获得了外源性AmpC酶基因后, Kp NTUH-K2044菌株AmpC酶诱导表型阳性,其相应的ampG基因缺失株AmpC酶诱导表型阴性.结论 成功构建肺炎克雷伯菌ampG基因缺失突变株,ampG基因缺失后肺炎克雷伯菌AmpC酶不能诱导表达.
Objective To construct mutant strains of Klebsiella pneumoniae with ampG gene dele-tion by homologous recombination and to evaluate the role of ampG gene in inducing the expression of AmpC enzyme.Methods Polymerase chain reaction （ PCR） was used to amplify the upstream and downstream fragments of ampG gene.The gene splicing by overlap extension PCR （ SOE-PCR） technique was used to construct the fusion fragment , which was then ligated into the temperature sensitive suicide vector pKO 3-km after enzyme digestion as pKO3-km-ΔampG.To achieve allelic exchange , the plasmid pKO3-km-ΔampG was introduced into Kp1 and Kp NTUH-K2044 strains by electroporation .The mutant strains of Klebsiella pneu-moniae with ampG gene deletion were screened out .The plasmid pACYC184-ampCR was introduced into the Kp NTUH-K2044 wild-type strain and its mutant strain with ampG gene deletion to make them harbor the gene encoding AmpC enzyme .The disk diffusion method was used to evaluate the effects of ampG gene on the expression of AmpC enzyme in Klebsiella pneumoniae strains with cefoxitin as the inducer .Results The recombinant plasmid pKO3-km-ΔampG was constructed successfully .The mutant strains of Klebsiella pneu-moniae with ampG gene deletion were constructed as verified by PCR and DNA sequencing .Compared with the Kp1 wild type strain, no AmpC enzyme was produced by the ampG gene knock-out Kp1 strain.The Kp NTUH-K2044 strain could produce AmpC enzyme , while the mutant strain of Kp NTUH-K2044 with ampG gene deletion could not after introduced the pACYC 184-ampCR plasmid .Conclusion The mutant strain of Klebsiella pneumoniae with ampG gene deletion was successfully constructed .The Klebsiella pneumonia strain without the ampG gene could not produce the AmpC enzyme .
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第10期723-728,共6页
Chinese Journal of Microbiology and Immunology
基金
黑龙江省自然科学基金研究项目（H2013100）
