Yoshihiro Megumi, Takamiya Osamu
Department of Biomedical Laboratory Sciences, Graduate School of Medicine, Health Sciences, Matsumoto 390-8621, Japan.
Rinsho Byori. 2012 Mar;60(3):193-200.
To investigate the influence of different transfected cells using expression studies of dysfunctional factor VII variants, we expressed recombinant FVII using COS-1 cells, CHO-K1 cells, BHK cells and HEK293 cells. The mutant type F7 DNAs with Leu-48Pro in the prepeptide domain, with Ser126Phe in the EGF-2 domain and with Gly354Cys in the catalytic domain were transfected in the four kinds of cell lines. Additionally, FVII Ser126Tyr and Ser126Gly were expressed to investigate different substitutions from Phe in position 126 in FVII. The FVII:Ag of recombinant FVII Leu-48Pro in conditioned medium, which were expressed in COS-1 cells, CHO-K1 cells, BHK cells and HEK293 cells, were 37.9% 68.0%, 11.7% and 9.2% respectively, when wild-type FVII was defined as 100%. The levels of FVII:Ag in cell lysates were 26.2%, 44.9%, 25.6% and 50.5%, respectively. The FVII:C of recombinant FVII Gly354Cys in conditioned medium were 3.7%, 3.5%, 1.5% and 1.7%, respectively. The levels of FVII:Ag were similar to FVII:C. The FVII:C of both recombinant FVII Ser126Phe and Ser126Tyr in conditioned medium of all four cell lines were considerably lower than that of FVII wild type. However, levels of FVII:Ag in conditioned medium of recombinant FVII S126G were 86.8% on COS-1 cells, 14.1% on CHO-K1 cells, 28.3% on BHK cells and 114.6% on HEK293 cells. When carrying out expression studies for FVII mutation, we have to consider different results based on the patient's laboratory phenotype when using different kinds of transfected cells.
为了通过功能失调的因子VII变体的表达研究来探究不同转染细胞的影响，我们使用COS-1细胞、CHO-K1细胞、BHK细胞和HEK293细胞表达重组FVII。将在前肽结构域具有Leu-48Pro、在EGF-2结构域具有Ser126Phe以及在催化结构域具有Gly354Cys的突变型F7 DNA转染到这四种细胞系中。此外，表达FVII Ser126Tyr和Ser126Gly以研究FVII中第126位苯丙氨酸的不同替代情况。当将野生型FVII定义为100%时，在COS-1细胞、CHO-K1细胞、BHK细胞和HEK293细胞中表达的重组FVII Leu-48Pro在条件培养基中的FVII:Ag分别为37.9%、68.0%、11.7%和9.2%。细胞裂解物中的FVII:Ag水平分别为26.2%、44.9%、25.6%和50.5%。重组FVII Gly354Cys在条件培养基中的FVII:C分别为3.7%、3.5%、1.5%和1.7%。FVII:Ag水平与FVII:C相似。所有四种细胞系的条件培养基中重组FVII Ser126Phe和Ser126Tyr的FVII:C均显著低于FVII野生型。然而，重组FVII S126G在条件培养基中的FVII:Ag水平在COS-1细胞上为86.8%，在CHO-K1细胞上为14.1%，在BHK细胞上为28.3%，在HEK293细胞上为114.6%。在进行FVII突变的表达研究时，使用不同类型的转染细胞时，我们必须根据患者的实验室表型考虑不同的结果。
