牛支原体武威分离株eno基因的克隆、表达及酶活测定  

Cloning, Expression and Determination of Enzymatic Activity of eno Gene from Mycoplasma bovis Wuwei Strain

作　　者：高翔[1] 王昱茜 邢小勇[1] 魏彦明[1] 包世俊[1] GAO Xiang;WANG Yu-Xi;XING Xiao-Yong;WEI Yan-Ming;BAO Shi-Jun(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《农业生物技术学报》2019年第5期936-942,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31360620);国家肉牛/牦牛产业技术体系项目(CARS-37)

摘　　要：牛支原体(Mycoplasma bovis, Mb)是引起肉牛(Bos taurus)和奶牛疾病的重要病原之一,给养牛业造成了重大的经济损失。烯醇化酶(enolase, eno)为糖酵解途径的关键限速酶,主要催化2-磷酸甘油酸转化为磷酸烯醇式丙酮酸。为研究Mb eno酶促活性及其动力学参数,本研究参照GenBank中Mb PG45株eno基因(NC_014760.1)序列设计特异性引物,应用PCR扩增获得Mb武威分离株的eno基因。在序列分析及基因优化的基础上,构建其原核表达载体p ET-eno并转化大肠杆菌(Escherichia coli)表达菌Transetta(DE3),经异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)诱导表达,在十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl dulfate polyacrylamide gel electropheresis, SDS-PAGE)分析的基础上,纯化表达产物对其酶促活性进行分析。结果表明,优化后的Mb武威株eno基因在大肠杆菌中成功获得表达,重组蛋白His-eno大小约为53 kD,且主要以可溶性蛋白形式存在;酶活分析结果显示:纯化产物His-eno的催化活性略高于兔肌肉烯醇化酶,且其最适温度为42℃,最适pH为8.0。双倒数作图所得Hiseno米氏常数(K_m)和最大反应速率(V_(max))分别为4.1×10^(-3)mol/L和3.4μmol/(L·min)。本研究结果为进一步研究Mb eno的生物学功能提供基础资料。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 (Km) and maximum reaction velocity (Vmax) 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.

关 键 词：牛支原体 烯醇化酶 原核表达 酶活性 

分 类 号：S852.62[农业科学—基础兽医学]