Molecular Mechenism of hemophilia B factor IXTaipei9

• Main Authors: Chia Ni Lin, 林佳霓
• Other Authors: Shu Wha Lin
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/86256313322441366657

碩士 === 國立臺灣大學 === 醫事技術學系 === 82 === B型血友病是一種隱性的性聯遺傳疾病，主要是因為病人血液中缺乏具有 凝血功能的第九因子，其發生率約每三萬到五萬個男性中有一位。本論文 以一個病人為對象，探討B型血友病病人致病的機轉以及第九因子蛋白結 構及功能的相關性。該病人為CRMr(cross reacting material reduced) ，其血液中第九因子含量為正常人的30-40%。經前人實驗的結果知其第九 因子基因上產生單點突變(point mutation)導致其第207個氨基酸由 Glycine變成Glutamic acid。實驗設計首先分離純化此病人之不正常第九 因子蛋白(以factor IXG207E表示)，再予以定性分析。經抽取病人血漿後 ，用DEAE-Sepharose CL-6B及anti-factor IX monoclonal antibody A7 兩種Column得以純化出第九因子，純化過程是以ELISA來定出第九因子蛋 白的含量。DEAE-Sepharose CL-6B column的回收率(recovery)為50%， A7 column的回收率為60%，總回收率為30%。得到的蛋白用銀染法及西方 點墨法(Western blot)確定。凝血功能的測試是用一階段行性部份凝血活 素時間(one stage Activated Partial Thromboplastin Time, aPTT)結 果顯示純化的factor IXG207E活性只有正常第九因子的3.5%。進一步分析 其生化活性得知factor IXG207E可以被factor XIa活化，當以鈣離子滴定 測試factor IXG207E與金屬離子結合能力時，可知其與正常第九因子非常 類似。然而當被第十一因子切割時，如加入活化中心探針 p- aminobenzamidine螢光劑，測定其active site conformation，發現 factor IXG207E無法釋放正常螢光，表示factor IXG207E的active site 可能不正常，以致無法結合p-aminobenzamidine。當利用純化的第十因子 作為受質，Ca2+，磷脂質與活化的factor IXG207E反應後，測定反應速率 為正常的1/40，表示該蛋白的酵素功能不正常。最後，本論文文建立一哺 乳細胞表達系統來探討CRMr的成因。利用 site directed mutagenesis， 得到含有該突變的cDNA，再利用哺乳細胞表達系統得到一轉感細胞株，以 製造factor IXG207E。以免疫沉澱法分析後發現正常的蛋白大部份分泌至 細胞外，而factor IXG207E比原型容易滯留在細胞內。因此推測CRMr的成 因之一可能是病人不易分泌出第九因子，所以造成病人的血液循還中的含 量比正常人少。 Factor IX Taipei 9 is factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules this variant contained a glycine to glutamic acid substitution at the 207th codon of mature factor IX. The molecular defect of factor IX Taipei 9 has been characterized in this study. Plasma derived factor IXG207E exhibited a mobility similar to that of normal factor IX on SDS-PAGE. Its specific activity was estimated to 3.5% of the normal factor IX in a one-stage activated partial thromboplastin time assay. Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence,both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E also binds antithrombin III equally as well as normal factor IXa. The functinal consequence of the mutated factor IX might result from the alternation of the microenvironment of the active site serine-365 inaccessible to its substrate. In addition, an in-vitro expression system has been set up for the expression of factor IXG207E in mammalian cells. A factor IX cDNA was mutated at the codon for the 207th amino acid by site directed mutagenesis and inserted into a mammalian expression vector pCMV5. The resultant plasmid was expressed in a human embryonic kidney cell line. Analysis of the cell extracts and media by immunoprecipitation followed by Western blotting has clearly demonstrated that, while the expressed wild type factor IX was secreted into the media, a large proportion of the factor IXG207E molecules was found inside the cells. The results suggest that the CRMr phenotype of factor IX Taipei 9 might be partly due to the retarded secretion of the mutated protein into