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Abstract
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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.
Language: zh |