Molecular Biological Analysis of Congenital Lactic Acidemia

丙酮酸脱氢酶α亚单位(E1α)缺乏症是先天性乳酸血症最常见的原因之一。这种缺陷的治疗通常是非常困难的。通过对突变型PDH蛋白功能的分子生物学分析，有望建立新的治疗方法。然后，在本项目中，我们首先尝试建立了正常和突变的推荐PDH蛋白的表达系统。构建了含有正常或突变的E1α和正常的E1β的表达载体，并将其导入大肠杆菌。在转化的大肠杆菌中表达了大量的PDH蛋白，纯化了推荐的PDH蛋白，并对其酶活性进行了测定。然而，正常推荐的PDH蛋白的酶活性远低于预期活性。该方法不能检测到几种突变的推荐PDH蛋白的酶活性，也不能检测到突变的PDH蛋白的功能障碍。因此，有必要建立一种构建具有足够酶活性的PDH蛋白的新方法。在本项目中，我们还建立了比我们以前建立的系统更安全、更快速的对酶学方法无法诊断的女性PDH缺乏症患者进行DNA诊断的系统。有了这个新的系统，4名女性患者可以被诊断为PDH缺乏症，这些患者用酶学方法无法诊断。

Pyruvate dehydrogenase (PDH) α-subunit (E1α) deficiency is one of the most common causes of congenital lactic acidemia. The therapy of this deficiency is usually very difficult. It is expected to establish the new therapy by molecular biological analysis of the function of mutant PDH protein. Then, in this project we first try to establish the expression system of normal and mutant recommbinat PDH proteins. The expression plasmid vector containing normal or mutant E1α and normal E1β cDNA was constructed and transfected into E. coli. In transformed E. coli, massive amount of PDH protein was expressed and the recommbinat PDH proteins was purified and the enzyme activity of recommbinat PDH protein was measured. However, the enzyme activity of normal recommbinat PDH protein was very lower than the expected activity. The enzyme activities of several mutant recommbinat PDH proteins could not be detected and the dysfunction of mutant PDH proteins could not detected by this method. Therefore, it is necessary to establish the new method for constrution of recommbinat PDH protein with enough enzyme activity.In this project, we also establish the more safe and faster new system of DNA diagnosis for female patients with PDH deficiency, who can not be diagnosed with enzymological method, than previously established system by us. With this new system, four female patients, who could not be daignased with enzymological method, could be diagnosed PDH deficiency.