摘要
Ldha基因表达可生成参与糖代谢的乳酸脱氢酶(LDH),通过克隆黄胸鼠Rattus tanezumi的Ldha基因并进行原核表达后可以研究其酶学性质。以黄胸鼠骨骼肌cDNA为模板,通过RT-PCR技术获得黄胸鼠Ldha基因编码序列,进行生物信息学分析后构建pET28α-Ldha重组表达质粒,导入大肠杆菌Escherichia coli BL21(DE3)中诱导表达,采用SDS-PAGE和Western Blot对表达蛋白进行鉴定,最后检测表达蛋白的酶学性质。结果显示,黄胸鼠Ldha基因编码区序列被成功克隆,纯化后的原核表达蛋白分子量约为37 kD,Western Blot证实后,分别以丙酮酸和乳酸为底物测得酶的平均比活性为113.760 U·mg^(-1)±1.463 U·mg^(-1)和2.180 U·mg^(-1)±0.125 U·mg^(-1),琼脂糖凝胶电泳同工酶谱分析显示该蛋白具有LDH活性且仅形成1条同工酶带。在pH7.0,25℃条件下,LDHA蛋白对丙酮酸、NADH、乳酸、NAD+4种底物的平均Km值分别为0.170 mmol·L^(-1)±0.193 mmol·L^(-1)、0.088 mmol·L^(-1)±0.008 mmol·L^(-1)、22.540 mmol·L^(-1)±0.007 mmol·L^(-1)、0.413 mmol·L^(-1)±0.069 mmol·L^(-1)。黄胸鼠Ldha基因编码序列被首次克隆并表达出具有活性的酶蛋白,分析了其酶学性质,可为后续研究啮齿类动物LDH的结构和功能提供基础资料。
The Ldha gene encodes a lactate dehydrogenase that involves in glucose metabolism.In this study,the Ldha gene of Rattus tanezumi was cloned and expressed,and its enzymatic properties was analyzed.The coding region of Ldha gene was obtained by RT-PCR using skeletal muscle cDNA of R.tanezumi.The recombinant expression vector pET28α-Ldha was constructed after bioinformatics analysis.The expressed protein was identified by SDS-PAGE and Western Blot.Finally,the enzymatic properties of the expressed product were measured.The results showed that the coding region of Ldha gene from R.tanezumi was successfully cloned and expressed with a product of 37 kD,as confirmed by Western Blot.By using pyruvate and lactic acid as the substances,the mean activities of the expressed protein product were 113.760 U·mg^(-1)±1.463 U·mg^(-1) and 2.180 U·mg^(-1)±0.125 U·mg^(-1),respectively.Agarose gel electrophoretic isoenzyme analysis showed that the protein had lactate dehydrogenase activity and formed only 1 isoenzyme band.Under the condition of pH7.0 and 25℃,enzymatic property analysis showed that the mean Km values to pyruvate,NADH,lactic acid and NAD+were 0.170 mmol·L^(-1)±0.193 mmol·L^(-1),0.088 mmol·L^(-1)±0.008 mmol·L^(-1),22.540 mmol·L^(-1)±0.007 mmol·L^(-1),and 0.413 mmol·L^(-1)±0.069 mmol·L^(-1),respectively.This study provides a basis for further study on the structure and function of lactate dehydrogenase in rodents.
作者
倪玉芳
张树军
方天星
万军
陈洁
曾凡才
NI Yufang;ZHANG Shujun;FANG Tianxing;WAN Jun;CHEN Jie;ZENG Fancai(Laboratory of Biochemistry and Molecular Biology, School of Basic Medical Science, Southwest Medical University, Luzhou, Sichuan Province 646000, China;Department of Nuclear Medicine, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan Province 646000, China)
出处
《四川动物》
北大核心
2021年第6期601-610,共10页
Sichuan Journal of Zoology
基金
西南野生动植物资源保护教育部重点实验室开放基金项目(XNYB17-3)
西南医科大学大学生创新创业训练计划项目(2020154)。
关键词
黄胸鼠
Ldha基因
原核表达
蛋白纯化
酶学性质
Rattus tanezumi
lactate dehydrogenase a(Ldha)gene
prokaryotic expression
protein purification
enzymatic property
