两种新突变导致血友病B的分子发病机制研究

Study on the molecular mechanism of two new mutations causing haemophilia B

目的 对所发现的2种FⅨ基因的新突变Cys82Ser和Ile288Ser进行体外研究,以探讨血友病B的分子发病机制.方法 克隆野生型PcDNA3.1（-）FⅨwt表达载体质粒,以此为模板,采用大引物法构建PcDNA3.1（-）FⅨM1（Cys82Ser）、PcDNA3.1（-）FⅨM2（Ile288Ser）突变表达载体质粒.采用脂质体法将这3种表达载体质粒分别瞬时转染人胚胎肾293细胞（HEK293细胞）和中国仓鼠卵巢细胞（CHO细胞）,同时以PcDNA3.1（-）空载体质粒为空白对照.经体外培养后获得相应的表达蛋白,所得产物分别采用ELISA法检测蛋白抗原 活性测定检测表达蛋白的FⅨ因子活性 免疫荧光染色法检测蛋白在细胞中的定位情况 RT-PCR法检测各培养细胞的FⅨmRNA水平.结果 2种突变体转染细胞的FⅨmRNA水平与野生型无明显区别.以PcDNA3.1（-）FⅨwt转染细胞裂解液和细胞培养上清液中的FⅨ：Ag为100.0%,细胞培养上清液中的FⅨ：C为100.0%,则PcDNA3.1（-）FⅨM1转染细胞裂解液和细胞培养上清液中的FⅨ：Ag分别为（99.4±4.1）%和（27.1±5.2）%,而细胞培养上清液中的FⅨ：C为（8.5±3.2）% PcDNA3.1（-）FⅨM2转染细胞裂解液和细胞培养上清液中的FⅨ：Ag分别为（31.7±2.5）%和（5.3±1.8）%,细胞培养上清液中的FⅨ：C＜1%.细胞免疫荧光试验也证实,转染PcDNA3.1（-）FⅨM1的CHO细胞,其荧光强度与转染PcDNA3.1（-）FⅨwt的CHO细胞相当,转染PcDNA3.1（-）FⅨM2的CHO细胞,其荧光强度明显弱于转染PcDNA3.1（-）FⅨwt的CHO细胞.结论 Cys82Ser突变由于改变了FⅨ的EGF-1区的分子空间构象从而可能导致突变蛋白分泌障碍并存在胞内滞留现象 Ile288Ser突变则可能导致突变蛋白分泌障碍或细胞内降解加速从而引起FⅨ活性降低.

Objective To study two new factor Ⅸ mutations Cys82Ser and Ile288Ser in vitro and research the molecular mechanism of haemophilia B. Methods PcDNA3. 1 （ - ） FⅨwt expression plasmid was prepared. The mutated FⅨcDNA expression plasmids, PcDNA3.1 （ - ） FⅨM1 （Cys82Ser） and PcDNA3. 1 （ - ） F Ⅸ M2 （Ile288Ser） were constructed by megaprimer method respectively. Transient expression experiments were performed using HEK293 cells transfected with the expression vectors containing the wild-type or the mutation recombinant cDNA. PcDNA3. 1 （ - ） was used as a blank control. The expression proteins were detected by ELISA, factor activity assay and flourescence stain. Results The results suggested that the two FⅨ gene mutations did not induce the reduction of the mutant FⅨ mRNA compared with the wild-type FⅨ mRNA. The FⅨ：Ag in culture media and cell lysate of wild type conduct were assigned as 100. 0%. The results of PcDNA3.1 （ - ） FⅨ M1 mutation protein were （27. 1 ± 5. 2）% and （99.4 ±4. 1）% respectively. For PcDNA3. 1（ - ）FⅨM2, the results were （5.3 ± 1.8）% and （31.7 ±2. 5）% respectively. The FⅨ： C in culture media of wild type conduct was also assigned as 100. 0%. Then the two types of mutant protein were （ 8. 5 ± 3.2 ） % and 〈 1%, respectively. Immunofluorescence microscopy result suggested that the intensity of perinuclear spot was reduced in cells transfected with PcDNA3.1 （ - ） FⅨM2 while staining for PcDNA3. 1 （ - ） FⅨM1 was predominantely diffuse without perinnclear enhancement. Conclusions These results strongly suggest that the FⅨ Cys82Ser mutation protein is not been correctly folded, by any possibility. The mutation protein has secretion defect. The secretion dysfunction and the protein degradation intracellular are possiblely the molecular pathology of Ile288Ser mutant protein.