China Animal Husbandry and Veterinary Medicine ›› 2020, Vol. 47 ›› Issue (1): 1-7.doi: 10.16431/j.cnki.1671-7236.2020.01.001

• Biotechnology • Previous Articles     Next Articles

Construction and Identification of Helicobacter hepaticus CdtB Gene Mutant Strain

ZHU Chen1,2, CAO Shuyang1,2, WU Zhihao1,2, YIN Jun1,2, ZHU Liqi1,2, ZHANG Quan1,2   

  1. 1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
    2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
  • Received:2019-08-02 Online:2020-01-20 Published:2020-01-17

Abstract: The experiment was aimed to knock out the CdtB gene of Helicobacter hepaticus (H.hepaticus).A suicide plasmid for knocking out the CdtB gene of H.hepaticus ATCC 51449 strain was constructed using the pcDNA plasmid.According to the principle of homologous recombination,the plasmid was transferred into H.hepaticus by electroporation to construct a H.hepaticus CdtB gene mutant strain (ΔCdtB).The ΔCdtB mutant strain was identified by PCR and sequencing.Using biochemical test and calculating the growth curve to detect the difference between the ΔCdtB mutant strain and the wild type H.hepaticus.The results showed that the homologous recombinant knockout plasmid pcDNA-ΔCdtb was constructed by plasmid pcDNA,and the chloramphenicol positive transformant was obtained by subculture and screening after the transformation.The PCR product of the mutant strain was 964 bp,which was larger than that of the corresponding product 884 bp of wild type H.hepaticus. It was found that the urease test results of ΔCdtB mutant strain and wild type strain were all positive,and there was no significant difference in growth rate between them on the common blood agar plate,but the ΔCdtB mutant strain grew more on the blood agar plate containing chloramphenicol.In addition,the deletion strain was passed on and identified by PCR,and no reverse mutation was found.To sum up,the gene knockout plasmid pcDNA-ΔCdtb constructed in this study could knock out the target gene of H.hepatica,and provide an effective gene manipulation tool for gene function research and pathogenic factor discovery of H.hepatica.

Key words: Helicobacter hepaticus; gene knockout; plasmid; homologous recombination

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