Molecular Biological Analysis of Congenital Lactic Acidemia

丙酮酸脱氢酶（PDH）α亚单位（E1α）缺乏是先天性乳酸血症最常见的病因之一。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。本项目首先尝试建立正常和突变型重组PDH蛋白的表达系统。构建了含有正常或突变型E1α和正常E1β cDNA的表达质粒载体，并转染E.杆菌转化E.大肠杆菌中大量表达重组PDH蛋白，并对重组PDH蛋白进行纯化和酶活性测定。然而，正常recommbinat PDH蛋白的酶活性远低于预期的活性。该方法不能检测几种突变型PDH蛋白的酶活性，也不能检测突变型PDH蛋白的功能障碍。因此，有必要建立一种新的酶活性的重组PDH蛋白的纯化方法，本课题还建立了一种新的DNA诊断系统，用于对酶法无法诊断的女性PDH缺乏症患者进行DNA诊断，比我们以前建立的系统更安全、更快速。应用该系统，对4例酶法检测不出的女性患者进行了PDH缺乏症的诊断。

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.