China Animal Husbandry and Veterinary Medicine ›› 2022, Vol. 49 ›› Issue (5): 1630-1640.doi: 10.16431/j.cnki.1671-7236.2022.05.004

• Biotechnology • Previous Articles     Next Articles

Construction and Biological Characteristics of Brucella Secreting Protein BMCO Gene Deletion Strain

QIU Runhui1, GUAN Feihu1, WANG Zihang1, GUO Jia1, ZHU Dexin1, ZHANG Wei1, WEI Chunyan1, HUO Mingkai1, SUN Zhihua1,2,3, ZHANG Hui1,2,3   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. Key Laboratory of Control and Prevention of Animal Disease, Xinjiang Production & Construction Corps, Shihezi 832000, China;
    3. State International Joint Research Center for Animal Health Breeding, Shihezi 832000, China
  • Received:2021-10-27 Online:2022-05-05 Published:2022-04-29

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Abstract: 【Objective】 The purpose of this study was to construct the multi copper oxidase (BMCO) gene deletion strain of Brucella bovis S2308,explore the growth characteristics of the deletion strain and its viability in host cells,and analyze the structure of BMCO protein.【Method】 The upstream and downstream homologous arms of BMCO gene and Kan gene were amplified by PCR,and the three genes were fused by fusion PCR.The fusion fragment was connected with pMD19-T vector to make Brucella bovis S2308 competent cells.1 800 V voltage and 400 Ω resistance were transferred to Brucella bovis S2308 competent cells and coated in Kan-resistant Brucella solid medium.Positive colonies were selected and cultured continuously for 10 generations.The positive genetic stability and growth trend of the 10th generation positive colonies were detected.The pBBR1MCS-4-BMCO fusion plasmid was electrotransferred into Brucella bovis which could stably inherit BMCO gene deletion,and the positive colonies were cultured and screened.The mouse macrophage RAW264.7 was infected with parent strain,deletion strain and supplementary strain with plural infection with MOI 100.The viability of the three strains in the cells was detected by plate counting method.【Result】 The results showed that the upstream and downstream homologous arms of BMCO gene and Kan gene with fragment size of 522,539 and 1 054 bp were successfully obtained,the recombinant vector of pMD19-T-BMCO-Kan fusion fragment was successfully constructed,the stable genetic deletion strain of BMCO gene was obtained,named S2308ΔBMCO,and the complementary strain of BMCO gene was successfully constructed,named ΔBMCO::BMCOS2308.The growth curve of the deleted strain was the same as that of the parent strain,which reached logarithmic growth phase at 12 h and entered the plateau phase at 30 h.After infecting mouse macrophage RWA264.7,compared with the parent strain S2308,the viability of the S2308ΔBMCO was extremely significantly decreased (P<0.01).Bioinformatics analysis showed that BMCO was a hydrophobic protein,located in the cytoplasm and contained a large number of random coil,alpha helix and extended chain,indicating that the protein had multiple binding sites.【Conclusion】 The deletion and replacement strains of BMCO gene of Brucella were successfully constructed.The deletion of BMCO gene did not affect its growth performance,but its viability in host cells decreased significantly.The structure of BMCO protein was preliminarily analyzed,which would lay a foundation for the further study of the function of protein secreted by Brucella.

Key words: Brucella; BMCO gene deletion strain; growth curve; intracellular survival

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