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黄胸鼠Ldha基因的克隆、原核表达及酶学性质研究
Cloning, Prokaryotic Expression and Enzymatic Properties of Ldha Gene from Rattus tanezumi
倪玉芳1, 张树军1, 方天星2, 万军1, 陈洁1, 曾凡才1*
点击:443次 下载:58次
DOI:10.11984/j.issn.1000-7083.20210055
作者单位:1. 西南医科大学基础医学院生物化学与分子生物学实验室, 四川泸州 646000;
2. 西南医科大学附属医院核医学科, 四川泸州 646000
中文关键字:黄胸鼠;Ldha基因;原核表达;蛋白纯化;酶学性质
英文关键字:Rattus tanezumi; lactate dehydrogenase a (Ldha) gene; prokaryotic expression; protein purification; enzymatic property
中文摘要: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 tanezumiLdha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。
英文摘要: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.
2021,40(6): 601-610 收稿日期:2021-02-19
分类号:Q78
基金项目: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)
作者简介:倪玉芳,女,硕士研究生,研究方向:生物化学与分子生物学,E-mail:1392782893@qq.com
*通信作者:曾凡才,男,博士,教授,研究方向:生物化学与分子生物学,E-mail:zfcai@swmu.edu.cn
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