[Establishment of a high expressing system of human coagulant factor VIII in vitro].
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.


CONCLUSIONS
The recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.
Cheng
Hai
H
Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Xu
Kai-Lin
KL
Sun
Hai-Ying
HY
Lu
Qun-Xian
QX
He
Xu-Peng
XP
Pan
Xiu-Ying
XY
D002460:Cell Line; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids; D014162:Transfection
D005169:Factor VIII