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[Retroviral-mediated high efficient in vitro expression of human coagulation factor VIII]
1Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Objective:
To develop a retroviral-mediated high efficient expression system of human coagulation factor VIII.
Methods:
The retroviral vector LNC-VIIIBD was generated by cloning a B-domain-deleted FVIII cDNA (760aa - 1639aa) into retroviral vector pLNCX. Several cell lines including NIH3T3, CHO, COS-7 and human hepatic cell line L-02 were infected with viral supernatant from the highest productive PA317 clones. The antigen and procoagulant activity of human FVIII in the cell culture medium were measured by ELISA assay and one-stage method, respectively. RT-PCR was performed for the detection of F VIII BD mRNA.
Results:
Human FVIII was expressed in all four target cells. The highest expression was observed in NIH3T3, the procoagulant activity of secreted FVIII was up to 1.6 U, and the FVIII antigen was 500 ng by 10(6) cells/ml in 24 hours, respectively.
Conclusion:
The constructed retroviral vector was able to generate high level expression of human FVIII in some cell lines, and it might have potential utility in the gene therapy for Hemophilia A.