›› 2019, Vol. 46 ›› Issue (8): 2257-2264.doi: 10.16431/j.cnki.1671-7236.2019.08.008

• Physiology and Biochemistry • Previous Articles     Next Articles

Effects of Ubiquitin-related Modified Protein SUMO-2 on the Survival of Brucella 16M in RAW264.7 Cells

DENG Xiaoyu1, YI Jihai1, LI Qifeng1, HE Jinke2, YANG Qin1, WANG Yueli1, WANG Zhen1, WANG Yong1, CHEN Chuangfu1   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. College of Life Sciences, Shihezi University, Shihezi 832000, China
  • Received:2019-03-21 Online:2019-08-20 Published:2019-08-17

Abstract:

To investigate the effect of ubiquitin-related modification protein SUMO-2 on Brucella 16M, RAW264.7 model of murine macrophages with SUMO-2 gene interference and overexpression was constructed and infected with Brucella 16M.Specific interference fragments and overexpression primers were designed according to the sequence of SUMO-2 gene in GenBank.After cloning, they were connected to the corresponding lentivirus vector and transformed into DH5α competent cells of Escherichia coli.HEK-293FT cells were transfected with plasmid extracted from positive clone bacteria, and RAW264.7 mouse macrophages were infected with recombinant lentivirus.The interference and overexpression cell models were successfully constructed by Brucella 16M infection, respectively.The transcription level of SUMO-2 was detected by Real-time PCR, the expression of SUMO-2 protein was detected by Western blotting, the levels of IFN-γ and TNF-α were detected by ELISA, and the viability of Brucella in cells was determined by colony count.The results showed that compared with control group, the level of SUMO-2 gene in interference group was extremely significantly lower (P<0.01), and the level of SUMO-2 gene in the overexpression group was extremely significantly higher (P<0.01).The cell model of SUMO-2 interference and overexpression was successfully constructed.Western blotting results showed that Brucella 16M infection could down-regulate the expression of SUMO-2 protein in a time-dependent manner.After colony counting, the number of Brucella extremely significantly decreased after SUMO-2 overexpression (P<0.01), which inhibited the intracellular reproduction of Brucella 16M.The number of Brucella significantly or extremely significantly increased after SUMO-2 interference (P<0.05;P<0.01), which promoted the intracellular reproduction of Brucella 16M.At the same time, the levels of IFN-γ and TNF-α extremely significantly increased after SUMO-2 overexpression (P<0.01).After SUMO-2 interference, the level of TNF-α significantly decreased (P<0.05), the level of IFN-γ extremely significantly decreased (P<0.01), and the expression of SUMO-2 in RAW264.7 cells also affected the production of IFN-γ and TNF-α.In conclusion, SUMO-2 protein played an important role in the survival of Brucella cells, which might help to elucidate the pathogenic mechanism of Brucella infection.

Key words: Brucella; SUMO-2; overexpression; interference; colony count

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