[Expression of B domain-deleted human coagulant factor VIII gene in 293T cells mediated by lentiviral vector in vitro].
This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.
Cheng
Hai
H
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221000, China. steve-cheng.china@yahoo.com.cn
Xu
Kai-Lin
KL
Sun
Hai-Ying
HY
DU
Bing
B
Zeng
Ling-Yu
LY
Lu
Qun-Xian
QX
He
Xu-Peng
XP
Pan
Xiu-Ying
XY
D002460:Cell Line; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D005169:Factor VIII