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Updated: Jun 21, 2026
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
[Establishment of a high expressing system of human coagulant factor VIII in vitro]
Hai Cheng1, Kai-Lin Xu, Hai-Ying Sun
1Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Objective:
To construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.
Methods:
BDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.
Results:
The infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.
Conclusion:
The recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.
