体外表达探讨凝血因子IXArg327Ile新突变导致血友病B的分子发病机制  

The molecular mechanism of haemophilia B caused by the Arg327Ile novel mutation in FIX gene by in vitro expression

作　　者：周佳维[1,2] 王鸿利[1] 戴菁[3] 郁婷婷[1] 陆晔玲[1] 丁秋兰[1] 王学锋[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院、上海血液学研究所医学基因组学国家重点实验室,200025 [2]现在浙江大学医学院附属第一医院,310003 [3]上海交通大学临床检验科

出　　处：《中华血液学杂志》2012年第8期642-647,共6页Chinese Journal of Hematology

基　　金：国家自然科学基金面上项目（30770904）;上海市教委科研创新项目（07zz39）

摘　　要：目的探讨凝血因子Ⅸ（FIX）Arg327Ile（R327I）突变导致血友病B的分子发病机制。方法在野生型FIX表达质粒FIX”peDNA3．1（一）的基础上，定点突变法构建FIXR327I、R327Ala（A）、R327Lys（K）和R327Asn（N）突变的表达质粒，重叠PCR构建FIX324～329（B折叠结构域）换成FVIl298～303同源结构突变表达质粒[FⅨBFVIIpeDNA3．1（一）]。将野生型及各种突变体表达质粒分别瞬时转染胚肾293T细胞，一期法检测培养上清液中FIX活性（FIX：C）及ELISA法检测细胞培养上清和细胞裂解液中FⅨ抗原（FⅨ：Ag），Westernblot法检测突变蛋白的相对分子质量及表达水平。荧光蛋白表达载体瞬时转染法检测活细胞中突变蛋白合成与分泌。结果体外瞬时转染FIXR327I突变基因的细胞FIX：C为正常野生型的4．49％，细胞上清液和细胞裂解液FIX：Ag水平分别为野生型的31．02％和129．29％，为交叉反应减低型（CRMR）。活细胞免疫荧光观察发现FIXR327I突变蛋白在细胞内含量较野生型显著增高，并且在细胞溶酶体内的含量较野生型也显著增多。FIXR327A、R327K、R327N及FIXl3FVII突变基因转染的细胞FIX：c水平较野生型减低，其中以FIXl3FVII突变降低最为明显；转染的细胞培养上清FIX：Ag水平除R327K突变型较野生型增加，其余突变型均比野生型有不同程度的降低。Westernblot法检测显示各种突变蛋白的相对分子质量与野生型相同而表达水平降低。结论FIXR327I突变基因可影响蛋白质合成和分泌，R327位点与FⅨ功能特异有关，其所在的B折叠结构域在FⅨ特异性功能中发挥重要作用。Objective To investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg32711e （R327I） in FIX gene. Methods The R327I, R327Ala（A） ,R327Lys（K） ,R327Asn （N） and a replacement mutant （FIX βFⅧ ） , in which FIXβ strand 324 -329 was replaced by that of F VII 298 - 303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type （WT） FIX expression plasmid. The HEK293 cell was transiently transfcct^d, then the activity of FIX （FIX： C） was assayed by one stage method in the conditioned medium, while the FIX： Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells. Results FIX： C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX： Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced（CRMR）. The viable cell flu- orescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX： C of the R327A, R327K, R327N and FⅪβFⅧ mutants was reduced compared to that of WT, the reduction of FIX： C of FIX J3FVII was the most significantly amount among all the mutants in medium. FIX： Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium. Conclusion The abnormal syn- thesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as th

关 键 词：血友病B 凝血因子IX 基因突变 瞬时表达 

分 类 号：R554.1[医药卫生—血液循环系统疾病]