凝血因子Ⅸ Arg327Ile突变蛋白功能缺陷机制研究

Studies on the mechanism of the Arg327Ile mutant causing defect in the function of factor Ⅸ

目的研究FⅨ Arg327Ile（R327I）及Arg327Ala（R327A）突变蛋白功能，探讨R327I突变导致血友病B的分子发病机制。方法采用潮霉素筛选稳定表达细胞株，ELISA法筛选高效表达FIX重组蛋白细胞株，采用快流速Q琼脂糖凝胶和离子交换两步法分离纯化重组蛋白。ELISA和SDS—PAGE电泳分别检测重组蛋白含量和纯度。FⅦa／TF／Ca“和FⅨa／Ca^2＋活化野生型及R327I和R327A突变FⅨ蛋白，用WB法分别检测不同时间内活化生成的FⅨa。野生型及两种突变FIXa在不同FⅧa浓度下活化激活FX，以此计算野生型及两种突变FⅨa与FⅧa的解离常数；在含或不含FⅧa的条件下，检测野生型及两种突变FⅨa对不同浓度FX的激活能力，以此计算其催化效率。结果表达分离纯化野生型、R327I和R327A FⅨ重组蛋白总量分别为450、210和64μg，SDS—PAGE电泳结果显示重组蛋白纯度符合要求。两种突变蛋白均能被FⅦa／TF／Ca^2＋和FⅪa／Ca^2＋正常激活。R3271与R327A两种FIXa突变蛋白与FⅧa的结合力分别为野生型的1／4和1／5。在含FⅧa条件下，R327I和R327I突变FⅨa对FX催化效率分别为野生型的1／6和1／8。而不含FⅧa条件下，催化效率分别为野生型的1／3和1／7．4。结论R327I和R327A两种突变FⅨa与FⅧa结合异常，R327位点参与FⅧa结合，同时也与底物活化相关。R327I突变与FⅧa结合力降低导致重型血友病B的发生.

Objective To investigate function of Arg327Ile （R327I） and Arg327Ala（R327A） FIX mutants and to study the molecular pathogenesis of haemophilia B（HB） caused by R327I mutation. Methods Hygromycin-resistant cell line was screened and the secretion of FIX antigen into the medium was measured by ELISA. The cell line with appropriate expression levels of FⅨ antigen was selected for culture. Recombinant FⅨ （rF Ⅸ ） was purified from concentrated medium by two step methods of Q- Sepharose Fast Flow and anion exchange chromatography. The concentration and purity of rF IX were determined by ELISA and SDS-PAGE, respectively. The activation of wild-type （WT）, R327I and R327A of rFⅨ by FⅦa/TF/Ca^2＋ or FXIa/Ca^2＋ was identified by Western blot in different time periods. The FIXa and FⅧa complex formed by interaction with different concentrations of FⅧa was used to activate F X, the apparent dissociation constant （Kd） for FⅧa binding was calculated by the kinetic results. The kinetic data of the activation of FX by WT, R327I and R327A FⅨa with or without FⅧa were calculated. Results The amount of WT, R327I and R327A rFIX were 450,210,64 μg,and the purity of rFⅨ was confirmed by SDSPAGE. Both R327I and R327A could be normally activated by FⅦa/TF/Ca^2＋ or FⅪa/Ca^2＋. Kd for FⅧa binding showed that the binding capacities of R327I and R327A were 4 and 5 times lower than WT, respectively. The catalytic efficiencies of R327I and R327A FⅨa for F X were 6 and 8 times lower with FⅧa, and 3 and 7.4 times lower without FⅧa, respectively. Conclusions R327I and R327A rF IX mutants impair their binding to the FⅧa. The site on R327 contributes to FⅧa binding. It is partly related to the activation of FX. The low FⅧa binding to R327I FIXa may cause HB.