牛支原体NOX2的原核表达及黏附特性

doi:10.11843/j.issn.0366-6964.2020.11.028

摘要/Abstract

摘要： 旨在探究牛支原体（Mycoplasma bovis，Mb）NADH氧化酶NOX2的生物学功能，本研究参照GenBank中Mb湖北分离株（Mb Hubei-1 strain）nox2基因序列设计引物，应用PCR扩增获得Mb临洮分离株的nox2基因，在测序及序列优化的基础上，构建原核表达载体pET-nox2，并在大肠杆菌Rosetta（DE3）中诱导表达，进而对表达产物rMbNOX2的酶促活性、免疫原性，NOX2在Mb内的分布，rMbNOX2抗血清的补体介导体外杀菌活性和对Mb黏附宿主细胞的抑制活性进行了分析。结果表明，Mb临洮株nox2基因全长1 350 bp，与GenBank中已知序列的Mb nox2基因序列比较，除Mb JF4278株相似性为97.93%外，其余均为99.93%。SDS-PAGE结果显示，优化的nox2基因在大肠杆菌中成功表达，重组蛋白rMbNOX2相对分子质量约为67 ku，且具有良好的酶促活性；ELISA与Western blot结果显示，rMbNOX2具有良好的免疫原性，且Mb NOX2在细胞浆中的分布多于细胞膜；补体介导的体外杀菌试验及黏附抑制试验证实，rMbNOX2抗血清具有明显的补体介导杀支原体活性，并可有效抑制Mb对宿主细胞的黏附。本研究为深入探讨Mb NOX2生物学功能奠定了基础。

关键词: 牛支原体, nox2基因, 原核表达, 功能分析

Abstract: To explore the biological function of NADH oxidase NOX2 from Mycoplasma bovis (Mb), according to the nox2 gene sequence of Mb strain Hubei (GenBank.CP002513.1), the primers were designed and the nox2 gene of Mb strain Lintao was amplified by PCR. Based on sequencing and gene optimization, the prokaryotic expression vector pET-nox2 was constructed, and was expressed in Escherichia coli Rosetta (DE3). Subsequently, the analysis of the enzymatic activity and the immunogenicity of recombinant proteins rMbNOX2 were completed. And then the subcellular localization of NOX2, complement-dependent bactericidal activity of anti-rMbNOX2 serum, and as well as inhibition effect of anti-rMbNOX2 serum to Mb adhering to host cells was determined. The results showed that the CDS sequence of the nox2 gene of Mb Lintao strain was 1 350 bp, and showed 99.93% homology with nox2 gene of all Mb except Mb JF4278 strain in GenBank. The result of SDS-PAGE displayed the optimized nox2 was successfully expressed in E. coli. The recombinant protein rMbNOX2 was about 67 ku. Enzyme activity analysis showed that the purified rMbNOX2 had good enzymatic activity. The results of ELISA and Western blot showed that the rMbNOX2 has excellent immunogenicity, and the Mb NOX2 distribute both in the cell membrane and cytoplasm, but it's more distributed in the cytoplasm. Complement-dependent mycoplasmacidal assay and adherence inhibition assay confirmed anti-rMbNOX2 serum has distinct complement-dependent mycoplasmacidal activity and can also effectively inhibit the adherence of Mb to host cells. The results of this study lay a foundation for further study on the biological function of Mb NOX2.

Key words: Mycoplasma bovis, nox2 gene, prokaryotic expression, function analysis

中图分类号:

S852.62

包世俊, 朱彩宏, 邢小勇, 丁小琴, 温峰琴, 武小椿, 薛惠文. 牛支原体NOX2的原核表达及黏附特性[J]. 畜牧兽医学报, 2020, 51(11): 2895-2902.

BAO Shijun, ZHU Caihong, XING Xiaoyong, DING Xiaoqin, WEN Fengqin, WU Xiaochun, XUE Huiwen. Prokaryotic Expression of NOX2 of Mycoplasma bovis and Its Adherence Characterization[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2895-2902.

参考文献 40

[1]	NICHOLAS R A J, AYLING R D. Mycoplasma bovis:disease, diagnosis, and control[J]. Res Vet Sci, 2003, 74(2):105-112.
[2]	HALE H H, HELMBOLDT C F, PLASTRIDGE W N, et al. Bovine mastitis caused by a Mycoplasma species[J]. Cornell Vet, 1962, 52:582-591.
[3]	ASKAA G, ERNØ H. NOTE:Elevation of Mycoplasma agalactiae subsp. bovis to Species Rank:Mycoplasma bovis (Hale et al.) comb. nov[J]. Int J Syst Evol Microbiol, 1976, 26(3):323-325.
[4]	PFÜTZNER H, SACHSE K. Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle[J]. Rev Sci Tech, 1996, 15(4):1477-1494.
[5]	胡长敏, 石磊, 龚瑞, 等. 牛支原体病研究进展[J]. 动物医学进展, 2009, 30(8):73-77.HU C M, SHI L, GONG R, et al. Progress on bovine Mycoplasmosis[J]. Progress in Veterinary Medicine, 2009, 30(8):73-77. (in Chinese)
[6]	辛九庆, 李媛, 郭丹, 等. 国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J]. 中国预防兽医学报, 2008, 30(9):661-664.XIN J Q, LI Y, GUO D, et al. First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(9):661-664. (in Chinese)
[7]	MENGHWAR H, HE C F, ZHANG H, et al. Genotype distribution of Chinese Mycoplasma bovis isolates and their evolutionary relationship to strains from other countries[J]. Microb Pathog, 2017, 111:108-117.
[8]	YAVLOVICH A, RECHNITZER H, ROTTEM S. α-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen[J]. Infect Immun, 2007, 75(12):5716-5719.
[9]	CHUMCHUA V, PORNPUTTAPONG N, THAMMARONGTHAM C, et al. Homology modeling of Mycoplasma pneumoniae enolase and its molecular interaction with human-plasminogen[J]. Bioinformation, 2008, 3(1):18-23.
[10]	CHEN H J, YU S Q, SHEN X Y, et al. The Mycoplasma gallisepticum α-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen[J]. Microb Pathog, 2011, 51(4):285-290.
[11]	SCHREINER S A, SOKOLI A, FELDER K M, et al. The surface-localised α-enolase of Mycoplasma suis is an adhesion protein[J]. Vet Microbiol, 2012, 156(1-2):88-95.
[12]	SONG Z Q, LI Y, LIU Y, et al. α-Enolase, an adhesion-related factor of Mycoplasma bovis[J]. PLoS One, 2012, 7(6):e38836.
[13]	BAO S J, GUO X Q, YU S Q, et al. Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J]. BMC Vet Res, 2014, 10(1):223.
[14]	QI J J, ZHANG F Q, WANG Y, et al. Characterization of Mycoplasma gallisepticum pyruvate dehydrogenase alpha and beta subunits and their roles in cytoadherence[J]. PLoS One, 2018, 13(12):e0208745.
[15]	GRÜNDEL A, FRIEDRICH K, PFEIFFER M, et al. Subunits of the pyruvate dehydrogenase cluster of Mycoplasma pneumoniae are surface-displayed proteins that bind and activate human plasminogen[J]. PLoS One, 2015, 10(5):e0126600.
[16]	SONG Z Q, LI Y, LIU Y, et al. α-Enolase, an adhesion-related factor of Mycoplasma bovis[J]. PLoS One, 2012, 7(6):e38836.
[17]	GUO Y P, ZHU H M, WANG J Y, et al. TrmFO, a Fibronectin-binding Adhesin of Mycoplasma bovis[J]. Int J Mol Sci, 2017, 18(8):1732.
[18]	GAO X, BAO S J, XING X Y, et al. Fructose-1, 6-bisphosphate aldolase of Mycoplasma bovis is a plasminogen-binding adhesin[J]. Microb Pathog, 2018, 124:230-237.
[19]	卿三红, 方柏山. NADH氧化酶的研究进展[J]. 华侨大学学报(自然科学版), 2011, 32(5):554-559.QING S H, FANG B S. Recent progress in research of NADH oxidase[J]. Journal of Huaqiao University (Natural Science), 2011, 32(5):554-559. (in Chinese)
[20]	JIANG R R, BOMMARIUS A S. Hydrogen peroxide-producing NADH oxidase (nox-1) from Lactococcus lactis[J]. Tetrahed Asymm, 2004, 15(18):2939-2944.
[21]	李林波, 罗宇, 屈凌波, 等. NADH氧化酶研究进展[J]. 河南工业大学学报(自然科学版), 2010, 31(4):80-88.LI L B, LUO Y, QU L B, et al. Research progress of NADH oxidase[J]. Journal of Henan University of Technology (Natural Science Edition), 2010, 31(4):80-88. (in Chinese)
[22]	李林波. 乳酸菌NADH氧化酶的筛选与基因克隆研究[D]. 郑州:河南工业大学, 2011.LI L B. Screening and gene cloning study of NADH oxidase from lactic acid bacteria[D]. Zhengzhou:Henan University of Technology, 2011. (in Chinese)
[23]	VAN DEN BERG G J, MCARDLE H J. A plasma membrane NADH oxidase is involved in copper uptake by plasma membrane vesicles isolated from rat liver[J]. Biochim Biophys Acta, 1994, 1195(2):276-80.
[24]	GE X C, SHI X L, SHI L M, et al. Involvement of NADH oxidase in biofilm formation in Streptococcus sanguinis[J]. PLoS One, 2016, 11(3):e0151142.
[25]	AKEDA Y. NADH oxidase, a new player in the field of Streptococcus suis infection[J]. Virulence, 2017, 8(1):11-12.
[26]	GE X C, YU Y, ZHANG M, et al. Involvement of NADH oxidase in competition and endocarditis virulence in Streptococcus sanguinis[J]. Infect Immun, 2016, 84(5):1470-1477.
[27]	YAN M G, YIN W B, FANG X, et al. Characteristics of a water-forming NADH oxidase from Methanobrevibacter smithii, an archaeon in the human gut[J]. Biosci Rep, 2016, 36(6):e00410.
[28]	ZHAO G, ZHANG H, CHEN X, et al. Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin[J]. Sci Rep, 2017, 7(1):44.
[29]	高翔, 邢小勇, 冯娜, 等. 牛支原体武威株fba基因的克隆、表达及亚细胞定位[J]. 农业生物技术学报, 2017, 25(2):274-281.GAO X, XING X Y, FENG N, et al. Cloning, expression and subcellular localization of fba gene in Mycoplasma bovis Wuwei strain[J]. Journal of Agricultural Biotechnology, 2017, 25(2):274-281. (in Chinese)
[30]	MEHINAGIC K, PILO P, VIDONDO B, et al. Coinfection of Swiss cattle with bovine parainfluenza virus 3 and Mycoplasma bovis at acute and chronic stages of bovine respiratory disease complex[J]. J Vet Diagn Invest, 2019, 31(5):674-680.
[31]	BÜRGI N, JOSI C, BURKI S, et al. Mycoplasma bovis co-infection with bovine viral diarrhea virus in bovine macrophages[J]. Vet Res, 2018, 49(1):2.
[32]	OLIVEIRA T E S, PELAQUIM I F, FLORES E F, et al. Mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows[J]. Transbound Emerg Dis, 2020, 67(S2):82-93.
[33]	BRAS A L, SULEMAN M, WOODBURY M, et al. A serologic survey of Mycoplasma spp. in farmed bison (Bison bison) herds in western Canada[J]. J Vet Diagn Invest, 2017, 29(4):513-521.
[34]	LYSNYANSKY I, FREED M, ROSALES R S, et al. An overview of Mycoplasma bovis mastitis in Israel (2004-2014)[J]. Vet J, 2016, 207:180-183.
[35]	REGISTER K B, JELINSKI M D, WALDNER M, et al. Comparison of multilocus sequence types found among North American isolates of Mycoplasma bovis from cattle, bison, and deer, 2007-2017[J]. J Vet Diagn Invest, 2019, 31(6):899-904.
[36]	GILLE L, CALLENS J, SUPRÉ K, et al. Use of a breeding bull and absence of a calving pen as risk factors for the presence of Mycoplasma bovis in dairy herds[J]. J Dairy Sci, 2018, 101(9):8284-8290.
[37]	POTHMANN H, SPERGSER J, ELMER J, et al. Severe Mycoplasma bovis outbreak in an Austrian dairy herd[J]. J Vet Diagn Invest, 2015, 27(6):777-783.
[38]	KLEIN U, DE JONG A, YOUALA M, et al. New antimicrobial susceptibility data from monitoring of Mycoplasma bovis isolated in Europe[J]. Vet Microbiol, 2019, 238:108432.
[39]	AUZAT I, CHAPUY-REGAUD S, LE BRAS G, et al. The NADH oxidase of Streptococcus pneumoniae:its involvement in competence and virulence[J]. Mol Microbiol, 1999, 34(5):1018-1028.
[40]	MUCHNIK L, ADAWI A, OHAYON A, et al. NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice[J]. PLoS One, 2013, 8(4):e61128.