泛素相关修饰蛋白SUMO-2对RAW264.7细胞内布鲁氏菌16M存活的影响

doi:10.16431/j.cnki.1671-7236.2019.08.008

摘要/Abstract

摘要：

为探究泛素相关修饰蛋白SUMO-2对布鲁氏菌16M的影响，本试验构建了SUMO-2基因干扰和过表达小鼠巨噬细胞RAW264.7模型，并用布鲁氏菌16M进行侵染。参照GenBank中SUMO-2基因序列设计特异性干扰片段和过表达引物，克隆成功后连接至相应的慢病毒载体，转化大肠杆菌DH5α感受态细胞，选取阳性克隆菌提取质粒转染HEK-293FT细胞，将重组的慢病毒感染小鼠巨噬细胞RAW264.7，利用布鲁氏菌16M分别侵染构建成功的干扰和过表达细胞模型。通过实时荧光定量PCR检测SUMO-2 mRNA的转录水平，Western blotting检测SUMO-2蛋白的表达，ELISA检测IFN-γ和TNF-α水平，菌落计数来确定布鲁氏菌在细胞中的存活能力。结果显示，与对照组相比，干扰组SUMO-2 mRNA水平极显著降低（P<0.01），过表达组SUMO-2 mRNA水平极显著升高（P<0.01），成功构建了SUMO-2干扰和过表达细胞模型。Western blotting结果显示，布鲁氏菌16M感染能以时间依赖的方式下调SUMO-2蛋白的表达。经菌落计数后发现，SUMO-2过表达后布鲁氏菌的数量极显著减少（P<0.01），抑制布鲁氏菌16M的细胞内繁殖。而SUMO-2干扰后布鲁氏菌的数量显著或极显著增加（P<0.05；P<0.01），促进布鲁氏菌16M的细胞内繁殖。同时，经SUMO-2过表达后，IFN-γ和TNF-α水平极显著升高（P<0.01）。经SUMO-2干扰后，TNF-α水平显著降低（P<0.05），IFN-γ水平极显著降低（P<0.01），SUMO-2在RAW264.7细胞中的表达变化也影响IFN-γ和TNF-α的产生。综上所述，SUMO-2蛋白在布鲁氏菌胞内存活中起着重要作用，可能有助于阐明布鲁氏菌感染的致病机制。

关键词: 布鲁氏菌; SUMO-2; 过表达; 干扰; 菌落计数

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

中图分类号:

S852.61

邓肖玉, 易继海, 李启峰, 何金科, 杨琴, 王月丽, 王震, 王勇, 陈创夫. 泛素相关修饰蛋白SUMO-2对RAW264.7细胞内布鲁氏菌16M存活的影响[J]. 《中国畜牧兽医》, 2019, 46(8): 2257-2264.

DENG Xiaoyu, YI Jihai, LI Qifeng, HE Jinke, YANG Qin, WANG Yueli, WANG Zhen, WANG Yong, CHEN Chuangfu. Effects of Ubiquitin-related Modified Protein SUMO-2 on the Survival of Brucella 16M in RAW264.7 Cells[J]. , 2019, 46(8): 2257-2264.

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