High level expression of human Factor Ⅷ in mammalian cells after retroviral-mediated gene transfer
10.3760/j.issn:0366-6999.2001.07.005
- VernacularTitle:逆转录病毒载体介导人凝血因子Ⅷ在哺乳细胞中的高效表达
- Author:
Xuemei GUO
1
;
Hongli WANG
;
HAIYAN CHU
;
Xuefeng WANG
;
Bin QU
;
Zhiguang LI
;
Zhengwu QI
;
Zhenyi WANG
Author Information
1. 上海瑞金医院
- Keywords:
retroviral vector;
Factor Ⅷ;
gene expression
- From:
Chinese Medical Journal
2001;114(7):690-693
- CountryChina
- Language:Chinese
-
Abstract:
Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.
