National Center for Clinical Laboratories, Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, PR China; Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, PR China.
National Center for Clinical Laboratories, Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, PR China.
Clin Biochem. 2021 May;91:45-51. doi: 10.1016/j.clinbiochem.2021.02.002. Epub 2021 Feb 20.
Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate inter-method bias and enhance equivalence among different measurement procedures, LDH detection needs to be standardized.
Optimized sequences coding for lactate dehydrogenase subunit A (LDH-A) and subunit B (LDH-B) were synthesized and cloned into the pRSFDuet vector, and then the constructed 6His-LDHA-pRSFDuet, 6His-LDHB-pRSFDuet, and 6His-LDHA-LDHB-pRSFDuet plasmids were transformed into Escherichia coli BL21 (DE3) for expression. The enzyme activity and specific activity of recombinant LDHs were detected. Electrophoresis of LDH isoenzymes was performed. The stability of recombinant LDHs and serum LDH was evaluated. Commutability of recombinant LDH-B was studied by the IFCC reference method and six routine methods.
Three plasmids were constructed and three highly concentrated recombinant LDH isoenzymes were obtained. The specific activities of LDH-A, LDH-AB, and LDH-B were 18.08 U/mg, 21.74 U/mg, and 14.18 U/mg, respectively. There was a desirable linear correlation between the activities of recombinant LDH isoenzymes and their protein concentrations. Electrophoresis of LDH isoenzymes showed that the recombinant LDH-B corresponded to LDH1 and it demonstrated good stability at 4 °C and 25 °C for 5 weeks. LDH-B formulations in saline-bovine serum albumin solution and human serum matrix were commutable for six routine methods.
Human recombinant LDH-B has great potential to become an improved and less expensive standard or reference material in external quality assessment for clinical LDH measurement.
乳酸脱氢酶（LDH）是一种关键酶，作为细胞损伤的标志物。其活性可通过各种实验室方法测量。为消除方法间的偏差并增强不同测量程序之间的等效性，需要对 LDH 检测进行标准化。
合成并克隆编码乳酸脱氢酶亚基 A（LDH-A）和亚基 B（LDH-B）的优化序列到 pRSFDuet 载体中，然后将构建的 6His-LDHA-pRSFDuet、6His-LDHB-pRSFDuet 和 6His-LDHA-LDHB-pRSFDuet 质粒转化到大肠杆菌 BL21（DE3）中进行表达。检测重组 LDH 的酶活性和比活性。进行 LDH 同工酶电泳。评估重组 LDH 和血清 LDH 的稳定性。通过 IFCC 参考方法和六种常规方法研究重组 LDH-B 的互换性。
构建了三个质粒，并获得了三种高浓度的重组 LDH 同工酶。LDH-A、LDH-AB 和 LDH-B 的比活性分别为 18.08 U/mg、21.74 U/mg 和 14.18 U/mg。重组 LDH 同工酶的活性与其蛋白浓度之间存在良好的线性相关性。LDH 同工酶电泳显示，重组 LDH-B 对应于 LDH1，并且在 4°C 和 25°C 下 5 周内具有良好的稳定性。生理盐水-牛血清白蛋白溶液和人血清基质中的 LDH-B 制剂与六种常规方法具有互换性。
人重组 LDH-B 具有成为临床 LDH 测量外部质量评估中改进且更经济的标准或参考物质的巨大潜力。
