Establishment of a high expressing system of human coagulant factor VIII in vitro / 中华血液学杂志
Chinese Journal of Hematology
;
(12): 166-170, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-314507
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Factor VIII
/
Transfection
/
Gene Expression
/
Cell Line
/
Lentivirus
/
Genetic Vectors
/
Genetics
/
Metabolism
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Hematology
Year:
2009
Type:
Article
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