Establishment Of Novel Lactate Assay Methods And Recombination Research Of Important Metabolic Enzymes

Energy metabolism is an important metabolism in organism,and the imbalance of energy metabolism is closely related to the occurrence and development of tumors.NAD(P)H-dependent dehydrogenases are key regulators of energy metabolism and play important roles in cell energy balance.Studies have confirmed abnormal expression and regulation of many NAD(P)H-dependent dehydrogenases in tumors,therefore reasearch on NAD(P)H-dependent dehydrogenases as anticancer drug targets has received increasing attention.Here we expressed,purified and assayed important NAD(P)H-dependent dehydrogenases involved in cellular energy metabolism.Among them,we focused on lactate dehydrogenase,and established a novel lactate dehydrogenase activity assay method and two new lactate determination methods by combining lactate dehydrogenase with NAD+/NADH sensor SoNar.Compared with traditional methods such as chromatography,the new methods were not only fast,easy to operate,but also low cost and high throughput.The limit of detection(LOD)and the limit of quantification(LOQ)for lactate were 2.60 ?M and 8.67 ?M,respectively.This method was very suitable for lactate determination range from 10 ?M to 1000 ?M.Besides,we constructed genetically encoded lactate sensors by fusing lactate dehydrogenase A with SoNar.These sensors were applied to the study of lactate metabolism in tumor cells.This thesis not only provides a valuable tool for basic life science research,but also provides innovative technology for drug screening of cancer and other metabolism diseases.