牛支原体0580基因C端截短体的原核表达及生物学功能初步分析

doi:10.11843/j.issn.0366-6964.2023.07.030

摘要/Abstract

摘要： 旨在初步分析牛支原体（Mycoplasma bovis） PG45菌株0580基因的生物学功能。前期测试发现牛支原体PG45菌株、08M菌株、07801菌株都有溶解鼠红细胞的现象，根据NCBI中对PG45菌株的基因注释，筛选到1个假定与溶血素相关的基因MBOVPG45_0580。该基因编码的蛋白经预测具有4个跨膜区，全长表达困难，故构建了不含跨膜区的C端截短体原核表达载体Pcold III-0580-C，进行诱导表达以及纯化，并在小鼠中制备0580-C端重组蛋白的多克隆抗体，对0580-C重组蛋白溶红细胞能力、多克隆抗体效价及特异性、是否影响菌株黏附细胞的能力进行了探究。PG45菌株中假定的与溶血素相关的蛋白0580在牛支原体中的相似性高达100%，去除4个跨膜区的重组蛋白0580-C能在大肠杆菌中表达，相对分子质量约为37 ku；鼠红细胞溶血试验发现0580-C重组蛋白没有溶血活性，但黏附及黏附阻断试验发现该蛋白有助于提高牛支原体黏附EBL细胞的效率，0580-C蛋白鼠源多抗能有效阻断牛支原体对EBL细胞的黏附。制备的0580-C蛋白鼠源多克隆抗体效价达2¹¹，且能识别到3株牛支原体菌株（PG45、08M、07801）天然表达的0580蛋白，表明制备的0580-C蛋白鼠源多克隆抗体特异性较好。牛支原体中假定的与溶血素相关蛋白0580可能是一种新的黏附相关分子，为解析牛支原体MBOVPG45_0580基因的生物学功能以及牛支原体致病机制提供了新的见解。

关键词: 牛支原体, 原核表达, 黏附作用, 0580-C重组蛋白

Abstract: The purpose of this study was to analyze the biological function of 0580 gene of Mycoplasma bovis (Mb) PG45 strain. In the previous stage, we found that PG45、08M and 07801 strain of M. bovis can lyse mouse red blood cells. So, according to the amino acid sequence of the putative hemolysin related gene 0580 in PG45 strain, gene MBOVPG45_0580 presumed to be related to hemolysin was screened. Due to the protein encoded by this gene is predicted to have four transmembrane regions and is difficult to express, we constructed the prokaryotic expression vector Pcold III-0580-C terminal fragment without transmembrane region to induce expression and purification, The recombinant protein 0580-C was immunized in BALB/c to prepare polyclonal antibody. At last, we explored whether the 0580-C recombinant protein has the ability of lysing RBCs and influences the ability of the strain to adhere to cell; Besides, the obtained polyclonal antibody specificity and titer were detected. The putative hemolysin protein 0580 in strain PG45 was 100% homologous to M. bovis, and the C-terminal truncated form of protein 0580 which removed four transmembrane regions could be successfully expressed in E. coli. The relative molecular weight of the purified recombinant 0580-C protein was about 37 ku. The hemolytic test of mouse RBCs found that the recombinant 0580-C protein has no hemolytic activity, but the cell adhesion test show that the protein contributes to improve the adhesion ability of Mb to EBL cell, polyclonal antibody of 0580-C protein could effectively block the adhesion process. The titer of the polyclonal antibody against 0580-C protein was as high as 2¹¹. The polyclonal antibody could recognize the 0580 protein expressed by three strains of M. bovis (PG45, 08M, 07801), which indicated that the polyclonal antibody against 0580-C protein was specific. This study found that the C-terminus of protein 0580 may be a novel adhesive-related molecule, which provides new insights into the biological function of putative hemolysin associated protein and the pathogenic mechanism of Mycoplasma bovis.

Key words: Mycoplasma bovis, prokaryotic expression, adhesion, 0580-C recombinant protein

中图分类号:

S852.62

荆婷婷, 尹号, 黄蓉, 兰仕梅, 李章程, 尤留超, 郝华芳, 付磊, 储岳峰. 牛支原体0580基因C端截短体的原核表达及生物学功能初步分析[J]. 畜牧兽医学报, 2023, 54(7): 2991-3001.

JING Tingting, YIN Hao, HUANG Rong, LAN Shimei, LI Zhangcheng, YOU Liuchao, HAO Huafang, FU Lei, CHU Yuefeng. Prokaryotic Expression and Biological Function Analysis of Mycoplasma bovis 0580 Gene C-terminal Truncated[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2991-3001.

参考文献 34

[1]	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.
[2]	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 pneumonia in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30 (9):661-664. (in Chinese)
[3]	郭雨丝, 陈颖钰, 赵刚, 等. 牛支原体病研究进展[J]. 中国奶牛, 2015(14):36-41.GUO Y S, CHEN Y Y, ZHAO G, et al. The progress on Mycoplasma Bovis[J]. China Dairy Cattle, 2015(14):36-41.(in Chinese)
[4]	The Veterinary Sciences Division of the Agri-Food and Biosciences Institute of Northern Ireland. Mycoplasma bovis pneumonia causing calf deaths in Northern Ireland[J]. Vet Rec, 2022, 191(4):e2151.
[5]	JUNQUEIRA N B, SALINA A, OLIVEIRA G C, et al. Detection of clinical bovine mastitis caused by Mycoplasma bovis in Brazil[J]. J Dairy Res, 2020, 87(3):306-308.
[6]	VALERIS-CHACIN R, POWLEDGE S, MCATEE T, et al. Mycoplasma bovis is associated with Mannheimia haemolytica during acute bovine respiratory disease in feedlot cattle[J]. Front Microbiol, 2022, 13:946792.
[7]	NICHOLAS R A. Bovine mycoplasmosis:silent and deadly[J]. Vet Rec, 2011, 168(17):459-462.
[8]	MAUNSELL F P, WOOLUMS A R, FRANCOZ D, et al. Mycoplasma bovis infections in cattle[J]. J Vet Intern Med, 2011, 25(4):772-783.
[9]	ASKAR H, CHEN S, HAO H, et al. Immune evasion of Mycoplasma bovis[J]. Pathogens, 2021, 10(3):297.
[10]	PEREZ-CASAL J. Pathogenesis and virulence of Mycoplasma bovis[J]. Vet Clin North Am Food Anim Pract, 2020, 36(2):269-278.
[11]	LIU Y, ZHOU M, XU S, et al. Mycoplasma bovis-generated reactive oxygen species and induced apoptosis in bovine mammary epithelial cell cultures[J]. J Dairy Sci, 2020, 103(11):10429-10445.
[12]	ADAMU J Y, WAWEGAMA N K, KANCI C A, et al. Mycoplasma bovis membrane protein MilA is a multifunctional lipase with novel lipid and glycosaminoglycan binding activity[J]. Infect Immun, 2020, 88(6):e00945-19.
[13]	ZHAO G, ZHU X, ZHANG H, et al. Novel secreted protein of Mycoplasma bovis MbovP280 induces macrophage apoptosis through CRYAB[J]. Front Immunol, 2021, 12:619362.
[14]	ZHANG H, ZHAO G, GUO Y, et al. Mycoplasma bovis MBOV_RS02825 encodes a secretory nuclease associated with cytotoxicity[J]. Int J Mol Sci, 2016, 17(5):628.
[15]	COLE B C, WARD J R, MARTIN C H. Hemolysin and peroxide activity of Mycoplasma species[J]. J Bacteriol, 1968, 95(6):2022-2030.
[16]	MINION F C, GOGUEN J D. Diffusion of target cell membrane proteins allows recognition of cryptic binding sites by Mycoplasma pulmonis hemagglutinin[J]. Infect Immun, 1985, 48(2):579-580.
[17]	ISHAG H Z A, XIONG Q Y, LIU M J, et al. Development of oriC-plasmids for use in Mycoplasma hyorhinis[J]. Sci Rep, 2017, 7(1):10596.
[18]	孙远航, 李敏, 王晶, 等. 绵羊肺炎支原体hlyA基因的表达及活性初步鉴定[J]. 农业生物技术学报, 2016, 24(3):435-442.SUN Y H, LI M, WANG J, et al. Expression and preliminary activity of hlyA gene from Mycoplasma ovipneumoniae[J]. Journal of Agricultural Biotechnology, 2016, 24(3):435-442. (in Chinese)
[19]	SOMERSON N L, PURCELL R H, TAYLOR-ROBINSON D, et al. Hemolysin of Mycoplasma pneumoniae[J]. J Bacteriol, 1965, 89(3):813-818.
[20]	ISHAG H Z A, XIONG Q Y, LIU M J, et al. E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis[J]. J Microbiol Methods, 2017, 136:49-56.
[21]	曹艳桃, 樊江峰, 余四九, 等. 牦牛EPF的原核表达及多克隆抗体制备[J]. 畜牧兽医学报, 2022, 53(2):470-480.CAO Y T, FAN J F, YU S J, et al. Prokaryotic expression of yaks EPF and its preparation of polyclonal antibodies[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2):470-480.(in Chinese)
[22]	ZOU X H, LI Y, WANG Y, et al. Molecular cloning and characterization of a surface-localized adhesion protein in Mycoplasma bovis Hubei-1 strain[J]. PLoS One, 2013, 8(7):e69644.
[23]	ZHU X, BARANOWSKI E, DONG Y, et al. An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis:Securing survival in cell culture[J]. PLoS Pathog, 2020, 16(6):e1008661.
[24]	张懿. 牛支原体PDHc-E2的原核表达及融合蛋白细胞黏附特性研究和单克隆抗体的制备[D]. 兰州:甘肃农业大学, 2018.ZHANG Y. Prokaryotic expression of the PDHc-E2 of Mycoplasma bovis and study on its adhesion property and preparation of monoclonal antibody[D]. Lanzhou:Gansu Agricultural University, 2018. (in Chinese)
[25]	SONG Z, LI Y, LIU Y, et al. α-enolase, an adhesion-related factor of Mycoplasma bovis[J]. PLoS One, 2012, 7(6):e38836.
[26]	胡政香, 孟庆玲, 乔军, 等. 绵羊肺炎支原体溶血素TlyC基因的克隆及分子特征分析[J]. 家畜生态学报, 2014, 35(9):18-22.HU Z X, MENG Q L, QIAO J, et al. Cloning and molecular characteristics of hemolysin TlyC gene from Mycoplasma Ovipneumoniae[J]. Journal of Domestic Animal Ecology, 2014, 35(9):18-22. (in Chinese)
[27]	ADAMU J Y, WAWEGAMA N K, BROWNING G F, et al. Membrane proteins of Mycoplasma bovis and their role in pathogenesis[J]. Res Vet Sci, 2013, 95(2):321-325.
[28]	XU Q Y, PAN Q, WU Q, et al. Mycoplasma Bovis adhesins and their target proteins[J]. Front Immunol, 2022, 13:1016641.
[29]	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.
[30]	HUANG J, ZHU H M, WANG J Y, et al. Fructose-1, 6-bisphosphate aldolase is involved in Mycoplasma bovis colonization as a fibronectin-binding adhesin[J]. Res Vet Sci, 2019, 124:70-78.
[31]	CHEN X, HUANG J, ZHU H M, et al. P27 (MBOV_RS03440) is a novel fibronectin binding adhesin of Mycoplasma bovis[J]. Int J Med Microbiol, 2018, 308(7):848-857.
[32]	WU X C, ZHANG S Y, LONG C Q, et al. Mycoplasmas bovis P48 induces apoptosis in EBL cells via an endoplasmic reticulum stress-dependent signaling pathway[J]. Vet Microbiol, 2021, 255:109013.
[33]	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.
[34]	包世俊, 朱彩宏, 邢小勇, 等. 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 S J, ZHU C H, XING X Y, et al. Prokaryotic expression of NOX2 of Mycoplasma bovis and its adherence characterization[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11):2895-2902. (in Chinese)