FUNCTIONAL CHARACTERIZATION OF HUMAN FACTOR VII BY MOLECULAR ANALYSIS OF INHERITED DYSFUNCTIONAL FACTOR VII VARIANT

1. 本文对来自12个无亲缘关系家庭的30例遗传性功能失调性因子7 （FVII）患者进行了研究。2个家族的先证者存在交叉反应物质阴性（CRM-）型缺陷，10个家族的先证者存在交叉反应物质阳性（CRM+）型缺陷。SSCP分析发现，与正常对照相比，在CRM+型外显子2中，每个人都有异常的迁移；一，外显子4；一，外显子5；一，外显子8；4 cases.3。用不同来源的组织因子分析了一例外显子4迁移异常的促凝活性。该病例在6055核苷酸位置发生G到A的转变，导致第1 egf样结构域的Arg 79被Gln取代，这与我们之前的报道相同（Biochem, 33: 14162-14169,1994）。在4例8外显子迁移异常的病例中，第一例在核苷酸位置11514发生C到T的跃迁，导致Thr359在催化域中被Met更多地取代，与文献报道一致（Blood, 89: 5085,1997）。第二例在11429核苷酸位置发生A向G过渡，导致Gly331被Ser取代，与先前报道相同（Blood Coag fibronl, 7: 93,1996）。第三例在11267核苷酸位置发生C到T的转变，导致Arg277被Cys取代。第4例在11487核苷酸位置发生T向G过渡，导致His348被Gln取代。利用Hep G2细胞的RNA，采用RT-PCR法制备FVII cDNA，表达各重组变体FVII。将野生型FVII cDNA插入表达载体pEE14中。转染CHO K1细胞。ELISA法检测培养基中FVII: ag。在培养基中加入维生素K孵育后，利用FVII耗尽血浆，用APTT法检测培养基中的FVII: c。利用寡核苷酸体外定点诱变技术，在野生型FVII cDNA序列中引入了每个密码子331,271和260的单碱基变化。少

1. Thirty cases from 12 unrelated families with inherited dysfunctional factor VII (FVII) were studies in order to investigate the function of factor VII.The probands from 2 families had a cross reactive material negative (CRM-) type deficiency, and the probands from 10 familie had a cross reactive material positive (CRM+) type deficiency.2. SSCP analysis identified an aberrant mobility relative to the normal control in each to the CRM+ type, exon 2 ; one, exon 4 ; one, exon 5 ; one, exon8 ; 4 cases.3. A case with an aberrant mobility in exon 4 was characterized by variable procoagulant activity using tissue factor from different sources. This case had G to A transition at nucleotide position 6055, which resulted in the substitution of Arg 79 by Gln in the 1st EGF-like domain, and was same case to our previous report (Biochem, 33 : 14162-14169,1994).4. In 4 cases with an aberrant mobility in exon 8, the first case had C to T transition at nucleotide position 11514, which resulted in th … More e substitution of Thr359 by Met in the catalytic domain, and was same to the previous report (Blood, 89 : 5085,1997). The 2nd case had A to G transition at nucleotide position 11429, which resulted in the substitution of Gly331 by Ser, and was same to the previous report (Blood Coag Fibrionl, 7 : 93,1996). The 3rd case had C to T transition at nucleotide position 11267, which resulted in the substitution of Arg277 by Cys. The 4th case had T to G transition at nucleotide position 11487, which resulted in the substitution of His348 by Gln.We prepared FVII cDNA by RT-PCR using RNA fron Hep G2 cell, in order to express each recombinant variant FVII.The wild-type FVII cDNA was inserted into the expression vector pEE14. CHO K1 cells were transfected. FVII : ag was detected in the medium by ELISA.After incuvated to add vitamin K into the medium, FVII : c was detected in the medium by APTT using FVII depleted plasma.6. A single base change at each codon 331,271 and 260 are introducing into the wild-type FVII cDNA sequence by oligonucleotide in vitro site-directed mutagenesis. Less