线粒体融合蛋白2基因干扰与过表达对布鲁氏菌诱导巨噬细胞凋亡的影响

doi:10.16431/j.cnki.1671-7236.2022.01.031

Abstract

Abstract: [Objective] The purpose of this experiment was to investigate the effect of mitochondrial fusion protein 2 (MFN2) gene interference and overexpression on mouse macrophages apoptosis induced by Brucella. [Method] Three siRNA sequences, siMFN2-450, siMFN2-1661, siMFN2-2275 and one negative interference sequence siMFN2-Negative specifically targeting MFN2 gene were designed by RNA interference techniques. A recombinant plasmid pcDNA3.1-EGFP-MFN2 was constructed by overexpression technique using empty plasmid pcDNA3.1-EGFP. The overexpression of recombinant plasmid pcDNA3.1-EGFP-MFN2 was identified by double enzyme digestion. The best interference sequence was screened and the overexpression efficiency of the recombinant plasmid was identified by Real-time quantitative PCR and Western blotting. The optimal interference sequence and the overexpressed recombinant plasmid were used to construct the macrophage model of MFN2 gene interference and overexpression, respectively. Macrophages were infected with Brucella bovis vaccine strain A19 for 24 hours according to the ratio of bacterial number to cell number (100:1). The expression of Bcl-2 related X protein (BAX) was detected by Real-time quantitative PCR and Western blotting, and cell apoptosis was detected by flow cytometry. [Result] The results of double enzyme digestion showed that the overexpression recombinant plasmid pcDNA3.1-EGFP-MFN2 was successfully constructed. Real-time quantitative PCR and Western blotting results showed that siMFN2-1661 could extremely significantly inhibit the expression of MFN2 (P<0.01), and pcDNA3.1- EGFP-MFN2 could extremely significantly enhance the expression of MFN2 (P<0.01). The transfected macrophage model with interference and overexpression of MFN2 gene was successfully constructed. After macrophages interfering with MFN2 gene expression were infected by Brucella, the protein expression levels of BAX was significantly higher than that in siMFN2-Negative group (P<0.05), transcription levels and the apoptosis rate of BAX were extremely significantly higher than that in siMFN2-Negative group (P<0.01). After macrophages overexpressing MFN2 gene were infected by Brucella, the transcription and protein expression of BAX were significantly lower than those in pcDNA3.1-EGFP group (P<0.05), and the apoptosis rate was extremely significantly lower than that in pcDNA3.1-EGFP group (P<0.01). [Conclusion] The study showed that MFN2 gene interference would enhance the ability of Brucella to induce cell apoptosis, while MFN2 gene overexpression would inhibit the ability of Brucella to induce cell apoptosis. The results could provide a reference for studying biological function of MFN2 gene and provide a theoretical basis for further analysis the pathogenic mechanism of Brucella.

Key words: mitochondrial fusion protein 2 (MFN2); Brucella; macrophages; apoptosis

CLC Number:

S828

XIE Shanshan, YANG Qin, DENG Xiaoyu, YI Jihai, WANG Zhen, CHEN Chuangfu. Effects of Mitochondrial Fusion Protein 2 Gene Interference and Overexpression on Macrophage Apoptosis Induced by Brucella[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 283-293.

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Effects of Mitochondrial Fusion Protein 2 Gene Interference and Overexpression on Macrophage Apoptosis Induced by Brucella

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