布鲁氏菌BspF蛋白生物信息学分析及其对巨噬细胞炎症因子表达的影响

doi:10.16431/j.cnki.1671-7236.2024.05.006

Abstract

Abstract: 【Objective】 The aim of this study was to analyze the bioinformatics of Brucella BspF protein,and further explore the effect of BspF protein on the expression of inflammatory cytokines in macrophages.【Method】 The physical and chemical properties,hydrophilicity and hydrophobicity,signal peptide,transmembrane region,antigenic determinant and domain,secondary structure and tertiary structure of Brucella BspF protein were analyzed by bioinformatics online software.Constructed recombinant plasmid pET-32a-BspF and then transformed into Escherichia coli BL21 (DE3) competent cells.BspF protein was induced by IPTG and purified.The expression and purification of BSPF protein were detected by SDS-PAGE and Western blotting.The toxicity of purified BspF protein on mouse macrophages (RAW264.7) was detected by CCK-8 method,and the effects of BspF protein on mRNA and protein expression of inflammatory factors in cells were detected by Real-time quantitative PCR and Western blotting,respectively.【Result】 The results showed that BspF protein,with a molecular mass of 48.75 ku,was an unstable protein with 15 antigenic determinant,no transmembrane region and no signal peptide,and the secondary structure was dominated by alpha helix.The prokaryotic expression vector of Brucella BspF gene was successfully constructed,and the target protein with the size of 60 ku was obtained after induced expression.Compared with control group,after the purified protein was stimulated at the final concentration of 50 μg/mL for 24 h,the mRNA expression of NLRP3,Caspase-1,IL-1β and IL-18 in BspF group were significantly or extremely significantly increased (P＜0.05 or P＜0.01),the protein expression of NLRP3 and Pro-Caspase-1 were extremely significantly increased (P＜0.01).【Conclusion】 Brucella BspF protein could trigger the inflammatory response of host cells through the NLRP3 inflammatome and promote the expression of inflammatory factors in macrophages,which provided a theoretical basis for the study of the inflammatogenic mechanism of Brucella and the screening of anti-inflammatory targets.

Key words: Brucella; BspF protein; bioinformatics; prokaryotic expression; inflammatory

CLC Number:

SUN Can, WEI Shuo, YAN Yujing, ZHAO Tianyi, ZHU Dexin, DENG Xingmei, GUO Jia, ZHANG Hui, SUN Zhihua. Bioinformatics Analysis of Brucella BspF Protein and Its Effect on Expression of Inflammatory Factors in Macrophages[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1846-1856.

References

[1] 徐朕宇,徐锦凤,邓肖玉,等.布鲁氏菌外膜囊泡生物信息学分析及免疫原性评价[J].中国畜牧兽医,2023,50(7):2906-2914. XU Z Y,XU J F,DENG X Y,et al.Bioinformatics analysis and immunogenicity evaluation of Brucella outer membrane vesicles[J].China Animal Husbandry & Veterinary Medicine,2023,50(7):2906-2914.(in Chinese)
[2] 文志,韩艳秋,王俊瑞.布鲁氏菌毒力因子研究进展[J].微生物学通报,2021,48(3):842-848. WEN Z,HAN Y Q,WANG J R.Virulence factors of Brucella:A review[J].Microbiology China,2021,48(3):842-848.(in Chinese)
[3] 袁莎,陈鹏博,陈创夫.布鲁氏菌LPS对小鼠巨噬细胞中NLRP3炎症小体转录水平的影响[J].中国畜牧兽医,2015,42(1):9-16. YUAN S,CHEN P B,CHEN C F.Effect of Brucella LPS on NLRP3 inflammasome transcriptional level of mouse macrophages[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):9-16.(in Chinese)
[4] 陶婷婷,赵天艺,李佳,等.TGF-β1在布鲁氏菌致炎过程中的作用及其与NLRP3炎症小体的相关性研究[J].动物医学进展,2022,43(2):48-53. TAO T T,ZHAO T Y,LI J,et al.Study on the role of TGF-β1 in Brucella induce inflammation and its correlation with NLRP3 inflammasome[J].Progress in Veterinary Medicine,2022,43(2):48-53.(in Chinese)
[5] 朱德馨,吴洁,郭嘉,等.布鲁氏菌BAB-RS25305蛋白对细胞炎症因子表达的影响[J].动物医学进展,2021,42(9):1-6. ZHU D X,WU J,GUO J,et al.Effect of BAB-RS25305 protein on expression of inflammatory cytokines[J].Progress in Veterinary Medicine,2021,42(9):1-6.(in Chinese)
[6] MILLER C N,SMITH E P,CUNDIFF J A,et al.A Brucella type Ⅳ effector targets the COG tethering complex to remodel host secretory traffic and promote intracellular replication[J].Cell Host & Microbe,2017,22:317-329.
[7] 邱润辉,关飞虎,王梓行,等.布鲁氏菌分泌蛋白BMCO基因缺失株的构建及生物学特性研究[J].中国畜牧兽医,2022,49(5):1630-1640. QIU R H,GUAN F H,WANG Z H,et al.Construction and biological characteristics of Brucella secrering protein BMCO gene deletion strain[J].China Animal Husbandry & Veterinary Medicine,2022,49(5):1630-1640.(in Chinese)
[8] BORGHESAN E,SMITH E P,MYENI A,et al.A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication[J].The European Molecular Biology Organization Journal,2021,40(19):e107664.
[9] LIN R,LI A,LI Y,et al.The Brucella effector protein BspF regulates apoptosis through the crotonylation of p53[J].Microorganisms,2023,11(9):2322.
[10] 马忠臣,胡瑞瑞,李芮芮,等.布鲁氏菌的胞内生存机制研究进展[J].动物医学进展,2021,42(6):71-78. MA Z C,HU R R,LI R R,et al.Progress on intracellular surivival mechanism of Brucella[J].Progress in Veterinary Medincine,2021,42(6):71-78.(in Chinese)
[11] ZHOU Y,BU Z,QIAN J,et al.The UTP-glucose-1-phosphate uridylyltransferase of Brucella melitensis inhibits the activation of NF-κB via regulating the bacterial type Ⅳ secretion system[J].International Journal of Biological Macromolecules,2020,164:3098-3104.
[12] XIONG X,LI B,ZHOU Z,et al.The VirB system plays a crucial role in Brucella intracellular infection[J].International Journal of Molecular Sciences,2021,22(24):13637.
[13] LUNDQUIST K,BILLINGS E,BI M,et al.The assembly of β-barrel membrane proteins by BAM and SAM[J].Molecular Microbiology,2021,115(3):425-435.
[14] 王书利,李梦含,高萌萌,等.布鲁氏菌分泌蛋白BPE043的功能及其对细胞凋亡基因表达的影响[J/OL].中国动物传染病学报,2022.Doi:10.19958/j.cnki.cn31-2031/s.20221207.003. WANG S L,LI M H,GAO M M,et al.Functional and effect on expressions of apoptosis-associated genes of Brucella type Ⅳ secretion system effector protein BPE043[J].Chinese Journal of Animal Infectious Diseases,2022.Doi:10.19958/j.cnki.cn31-2031/s.20221207.003.(in Chinese)
[15] 陈蕾,张广冻,李俊玫,等.布鲁氏菌入侵相关基因IalB缺失株的构建及生物学特性分析[J].微生物学报,2023,63(3):1254-1268. CHEN L,ZHANG G D,LI J M,et al.Construction and characterization of IalB-deleted strain of Brucella suis[J].Acta Microbiologica Sinica,2023,63(3):1254-1268.(in Chinese)
[16] 杨江流,乔军伟,宋青山,等.布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析[J].中国畜牧兽医,2021,48(8):2746-2753. YANG J L,QIAO J W,SONG Q S,et al.Prokaryotic expression and bioinformatics analysis of virulence gene BvfA of Brucella S19[J].China Animal Husbandry & Veterinary Medicine, 2021,48(8):2746-2753.(in Chinese)
[17] 肖洋洋,马忠臣,李芮芮,等.布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备[J].中国畜牧兽医,2022,49(8):3112-3121. XIAO Y Y,MA Z C,LI R R,et al.Bioinformatic analysis and polyclonal preparation of Brucella secreted protein BspI[J].China Animal Husbandry & Veterinary Medicine,2022,49(8):3112-3121.(in Chinese)
[18] 蒋丹露,刘阳阳,孙如愚,等.NLRP3炎症小体介导的炎症相关疾病研究进展[J].生命科学,2017,29(9):898-907. JIANG D L,LIU Y Y,SUN R Y,et al.NLRP3 inflamamasome and its role in inflammation related diseases[J].Chinese Bulletin of Life Sciences,2017,29(9):898-907.(in Chinese)
[19] JO E K,KIM J K,SHIN D M D,et al.Molecular mechanisms regulating NLRP3 inflammasome activation[J].Cellular & Molecular Immunology,2016,13(2):148-159.
[20] BRONNER D N,ABUAITA B H,CHEN X,et al.Endoplasmic reticulum stress activates the inflammasome via NLRP3-Caspase-2 driven mitochondrial damage[J].Immunity,2015,43:451-462.
[21] LOB S,AMANN N,KUHN C,et al.Interleukin-1 beta is significantly upregulated in the decidua of spontaneous and recurrent miscarriage placentas[J].Journal of Reproductive Immunology,2021,144:103283.

Bioinformatics Analysis of Brucella BspF Protein and Its Effect on Expression of Inflammatory Factors in Macrophages

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZOU Juhong, MO Zhihua, LIU Yufan, ZOU Jianwei, LU Jun, HE Haien, WANG Fan, HUANG Yanna, JIANG Qinyang. Cloning,Bioinformatics Analysis and Eukaryotic Expression Vector Construction of Rheb Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1819-1826.
[2]	PAN Shiyu, YIN Na, WANG Jiaxing, WANG Qiangzhou, CHEN Shihao, BAI Hao, CHANG Guobin. Bioinformatic Analysis of Chicken DCAF1 and Its Impact on the Replication of ALV-J [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1837-1845.
[3]	CAI Chang, CHEN Hewei, SONG Ruipeng, QIN Shaomin, LIU Jinfeng, QIN Shuying, SUN Qian, CHEN Tongjinyue, OU Changbo, LIU Xingyou, MA Ling, WU Jianmin. Bioinformatics Analysis and Preparation of Polyclonal Antibody of NSs Protein of Novel Akabane Virus [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2015-2026.
[4]	BAO Hua, HU Chunli, AN Yanhao, ZHANG Chunhua, MA Yanfen. Effects of EGCG on Inflammatory Injury and Apoptosis of BMECs Induced by LPS [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2122-2131.
[5]	WANG Yan, ZHANG Ying, ZHAO Yani, YI Lin, SHI Zhen, ZHOU Changming, ZHOU Wanrong, JIANG Lili, FAN Zhaobin. Cloning,Bioinformatics and Tissue Expression Characteristics of STING Gene in White Leghorn Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1372-1381.
[6]	YAN Zunqiang, LIANG Yuhao, SONG Kelin, GUN Shuangbao, WANG Pengfei. Cloning,Bioinformatics Analysis and Tissue Expression of SIRT3 Gene in Hezuo Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1390-1399.
[7]	SONG Qingshan, YANG Jiangliu, LIU Jun, JIA Fang. Multi-omics Analysis of the Effect of Brucella Virulence Factor A on the NADH-ubiquinone Oxidoreductase Complex Ⅰ of Mitochondria in Sertoli Cells [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1400-1409.
[8]	MA Rong, LA Yongfu, BAO Pengjia, GUO Xian, LU Jianwei, ZHA Lao, ZHAO Xue, LIANG Chunnian, CHENG Shuru. Cloning,Bioinformatics Analysis and Tissue Expression of MYL9 Gene in Meiren Yak [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1410-1419.
[9]	YIN Siqi, ZHAO Gangkui, GAO Haixu, SUN Honghong, XIN Yiran, ZAN Linsen, ZHAO Chunping. Cloning, Bioinformatics Analysis and Tissue Expression of FADS Gene Family in Qinchuan Cattle [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 903-915.
[10]	SONG Xingchao, MENG Jinzhu, ZHAO Yuanyuan, WU Zhenyang, AN Qingming. Cloning and Bioinformatics Analysis of myogenin Gene in Guizhou White Goat [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 926-935.
[11]	ZHOU Yingqing, LIU Yuxuan, YE Zhiwang, PEI Xinru, DU Xin, GAO Yang. Development of Squalene and Its Application in Animal Production [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 1077-1085.
[12]	REN Jianle, LIN Yiting, JI Kang, TAN Shanshan, CHEN Xinxin, JIN Yi, WANG Ying, NIU Sheng, LIANG Libin, LI Junping, ZHAO Yujun, TIAN Wenxia. Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 678-688.
[13]	LIANG Zhixuan, MEI Chen, ZHANG Xue, XU Haojun, ZHI Yan, LI Huanrong, WANG Hongjun. Prokaryotic Expression and Immunogenicity of lpxM Protein of Avibacterium paragallinarum [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 719-727.
[14]	TANG Xinggang, LUO Shengjun, YUAN Minggui, TIAN Ya, SHU Bingyao, YANG Xi, LI Yuelong, XIANG Rong. Study on Therapeutic Effect of Aqueous Extract of Vitex negundo L.var.cannabifolia (Sieb.et Zucc.) Hand.-Mazz.on Castor Oil Induced Diarrhea in Mice [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 799-808.
[15]	LIU Qi, LIU Zhengwei, LIANG Lin, ZHANG Li, HAO Chunhui, LI Yue, ZHAO Fuqing. Amplification and Bioinformatics Analysis of VP2 Gene of a Liaoning Shenyang Strain of Feline Panleukopenia Virus [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 23-32.