|Budget Amount *help
¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1997 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1996 : ¥1,000,000 (Direct Cost : ¥1,000,000)
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
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.
3、wild typeの第VII因子発現と同様に発現ベクター〓4GSに導入すべく、塩基配列の分析によって得られた成績から変異型DNAを作製中である。 Less