牛支原体武威分离株<em>eno</em>基因的克隆、表达及酶活测定

doi:10.3969/j.issn.1674-7968.2019.05.017

Abstract
Figure/Table
References
Related Citation (15)

Download: PDF (1575 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Mycoplasma bovis (Mb) is one of the most important pathogens causing diseases in beef cattle (Bos taurus) and cow, which cause great economic loss to cattle industry.Enolase (eno) is the key rate-limiting enzyme in glycolysis pathway and catalyzes the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP).In order to study the enzymatic activity of Mb eno, the specific primers were designed according to the eno gene sequence of Mb PG45 strain in GenBank (NC_014760.1) and the eno gene of Mb Wuwei strain was amplified by PCR.Based on the sequence analysis and the gene optimization, the prokaryotic expression vector pET-eno was constructed and transformed into Escherichia coli Transetta (DE3).Then the recombinant protein was expressed with induction by isopropyl β-D-1-thiogalactopyr-Anoside (IPTG).Subsequently, the expression product was purified and the assay of its enzymatic activity was completed.The results showed that the optimized eno gene of Mb Wuwei strain was successfully expressed in DE3 in soluble form and the relative molecular weight of the recombinant protein His-eno was approximately 53 kD.The results of enzymatic activity analysis indicated that the catalytic activity of His-eno was slightly higher than that of rabbit muscle enolase, the optimum temperature was 42 ℃ and the optimal pH was 8.0.The michaelis constant (K_m) and maximum reaction velocity (V_max) were 4.1×10^-3 mol/L and 3.4 μmol/(L·min), respectively.The results of this study could provide basic data for further study on the biological function of Mb eno.

Key words： Mycoplasma bovis Enolase Prokaryotic expression Enzymatic activity

Received: 28 September 2018

ZTFLH:

S852.62

Corresponding Authors: bsjdy@126.com

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	GAO Xiang
	WANG Yu-Xi
	XING Xiao-Yong
	WEI Yan-Ming
	BAO Shi-Jun

Cite this article:

GAO Xiang,WANG Yu-Xi,XING Xiao-Yong, et al. Cloning, Expression and Determination of Enzymatic Activity of eno Gene from Mycoplasma bovis Wuwei Strain[J]. 农业生物技术学报, 2019, 27(5): 936-942.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2019.05.017 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2019/V27/I5/936

[1] 陈嘉棣, 黎济申, 张赋平, 等.1983.上海乳牛霉形体的分离与鉴定[J].畜牧兽医学报, 14(4): 277-283.
(Chen J D, Li J S, Zhang F P, et al.1983.Isolate and identification of Mycoplasma from dairy cattle[J].Chinese Journal of Animal and Veterinary Science, 14(4): 277-283.)
[2] 高翔, 邢小勇, 冯娜, 等.2017.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, 25(2): 274-281.
(Gao X, Xing X Y, Feng N, et al.2017.Cloning, expression and subcellular localization of fba gene in Mycoplasma bovis Wuwei Strain[J].Journal of Agriculture Biotechnology, 25(2): 274-281.)
[3] 韩猛立, 康立超, 钟发刚, 等.2013.细菌性牛呼吸道疾病的研究进展[J].中国畜牧兽医, 40(5): 165-172.
(Han M L, Kang L C, Zhong F G, et al.2013.Research progress on bacterial pathogens of the bovine respiratory disease[J].China Animal Husbandry & Veterinary Medicine, 40(5): 165-172.)
[4] 沈欣悦.2013.鸡毒支原体两种相关酶的生物学功能研究[D].硕士学位论文, 扬州大学, 导师: 丁铲, 秦爱建, pp.2.
(Shen X Y.2013.Studies on biological functions of two membrane-associated enzymes in Mycoplasma gallisepticum[D].Thesis for M.S.Yangzhou Agriculture University, Supervisor: Ding C, Qin A J, pp.2.)
[5] 吴移谋, 叶元康.2008.支原体学[M].北京: 人民卫生出版社, pp.3-4.
(Wu Y M, Ye Y K.2008.Mycoplasmology[M].Beijing: The People's Medical Press.pp.3-4.)
[6] 辛九庆, 李媛, 郭丹, 等.2008.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, 30(9): 661-664.
(Xin J Q, Li Y, Guo D, et al.2008.First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J].Chinese Journal of Preventive Veterinary Medicine, 30(9): 661-664.)
[7] Bao S J, Guo X Q, Yu S Q, et al.2014.Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J].BMC Veterinary Research, 10(1): 223-232.
[8] Burki S, Frey J, Pilo P.2015.Virulence, persistence and dissemination of Mycoplasma bovis[J].Veterinary Microbiology, 179(1-2): 15-22.
[9] Chen H J, Yu S Q, Shen X Y, et al.2011.The Mycoplasma gallisepticum α-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen[J].Microbiology Pathogenesis, 51(4): 285-290.
[10] Daubenspeck J M, Liu R, Dybvig K, 2016.Rhamnose links moonlighting proteins to membrane phospholipid in Mycoplasmas[J].PLoS One, 11(9): e0162505.
[11] Fontan P A, Pancholi V, Nociari M M, et al.2000.Antibodies to streptococcal surface enolase react with human α-enolase: Implications in poststreptococcal sequelae[J].Journal of Infectious Diseases, 182(6): 1712-21.
[12] Hale H H, Helmboldt C F, Plastridge W N, et al.1962.Bovine mastitis caused by a Mycoplasma species[J].The Cornell Veterinarian, 52: 582-591.
[13] Maunsell F P, Woolums A R, Francoz D, et al.2011.Mycoplasma bovis infections in cattle[J].Journal of Veterinary Internal Medicine, 25(4): 772-783.
[14] Nicholas, R A.2011.Bovine mycoplasmosis: Silent and deadly[J].Veterinary Record, 168(17): 459-462.
[15] Pancholi V.2001.Multifunctional alpha-enolase: Its role in disease[J].Cellular and Molecule Life Science, 58(7): 902-920.
[16] Rasheed M A, Qi J, Zhu X, et al.2017.Comparative genomics of Mycoplasma bovis strains reveals that decreased virulence with increasing passages might correlate with potential virulence-related factors[J].Frontiers in Cellular and Infection Microbiology, 7(5): 177-191.
[17] Song Z Q, Li Y, Liu Y, et al.2012.α-Enolase, an adhesion-related factor of Mycoplasma bovis[J].PLoS One, 7(6): e38836.
[18] Whiting G C, Evans J T, Patel S, et al.2002.Purification of native a-enolase from Streptococcus pneumoniae that binds plasminogen and is immunogenic[J].Journal of Medical Microbiology, 51(10): 837-843.
[19] Vega-Rodriguez J, Ghosh A K, Kanzok S M, et al.2014.Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut[J].Proceedings of the National Academy of the USA, 111(4): E492-E500.