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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