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目的探讨F9基因Arg327Ile(R327I)突变导致血友病B的分子发病机制研究。方法对1名血友病B患者作实验室和基因诊断,定点突变法构建F9基因R327I突变的表达质粒;瞬时转染HEK293细胞,一期法检测细胞上清液中凝血因子Ⅸ活性(FⅨ∶C),ELISA法检测细胞上清液和裂解中FⅨ抗原(FⅨ∶Ag),Western blotting检测R327I突变蛋白的分子量及表达量;免疫荧光共定位染色法检测突变蛋白在内质网和高尔基体内分布。结果瞬时表达显示该患者突变细胞标本上清液中R327I突变型FⅨ∶C为野生型的4.49%,明显降低;细胞上清液和裂解液FⅨ∶Ag分别为野生型的31%和129%,为交叉反应物质减低型(CRMR)。Western blotting显示细胞上清液中R327I突变蛋白分子量与野生型相同,但含量比野生型明显降低;免疫荧光共定位染色显示R327I突变蛋白在内质网中分布较野生型多,而在高尔基体中较野生型少。结论 F9基因R327I突变影响蛋白质合成和分泌,突变蛋白较野生型表达量偏低,同时R327I突变蛋白存在凝血功能缺陷从而导致血友病B。
Objective To investigate the molecular pathogenesis of hemophilia B caused by Fg gene Arg327Ile (R327I) mutation. Methods One hemophilia B patient was diagnosed by laboratory and gene diagnosis. The R327I mutation expression plasmid of F9 gene was constructed by site-directed mutagenesis. HEK293 cells were transiently transfected and the activity of coagulation factor IX C). The supernatant and the FIX antigen (FIX: Ag) were detected by ELISA. The molecular weight and expression of R327I mutant protein were detected by Western blotting. The distribution of mutein in the endoplasmic reticulum and Golgi was detected by immunofluorescence co-localization staining . Results Transient expression showed that the R327I mutant FIX:C in the supernatant of the mutant cell was 4.49% of the wild type, which was significantly decreased. The supernatant and lysate FⅨ:Ag were 31% and 129% Cross-Reactive Substances Reduced (CRMR). Western blotting showed that the molecular weight of the R327I mutant in the cell supernatant was the same as that of the wild type but significantly lower than that of the wild type. Immunofluorescence co-localization staining showed that R327I mutein was more distributed in the endoplasmic reticulum than in the wild type, Less than the wild type. Conclusion The R327I mutation of F9 gene affects the protein synthesis and secretion, while the mutein is lower than the wild type. Meanwhile, the R327I mutant protein has coagulation defects, leading to hemophilia B.