B deletion of the human clotting factor Ⅷ gene in 293T cells.docVIP

 PAGE \* MERGEFORMAT 24 B deletion of the human clotting factor Ⅷ gene in 293T cells Authors: Chenghai, Xu Kai-lin, SUN Hai-Ying, Du Bing, Zeng Yu, deer first, He Xu Peng, Xiu-Ying Pan Abstract The purpose of this study is built containing human clotting factor Ⅷ (F Ⅷ) gene lentiviral vector was observed in 293T cells expression. With the restriction enzyme to obtain the missing B, human clotting factor Ⅷ gene (BDDhF Ⅷ cDNA) fragments to be cloned lentiviral vector pXZ208, constructed lentiviral vector pXZ208  BDDhF Ⅷ; with the restriction enzyme identification of carrier the connection direction, using calcium phosphate coprecipitation method will pXZ208  BDDhF Ⅷ recombinant plasmid and packaging plasmids were ΔNRF, coated tablets VSV  G proteins were co-transfected 293T packaging cells, packaged infected 293T cells, and to pXZ171 as a control . After infection by reverse transcription  polymerase chain reaction (RT  PCR) detection of BDDhF Ⅷ gene transcription, a cell culture supernatant was detected F Ⅷ activity, flow cytometry (FCM) detection of vector infection efficiency, PCR detection of BDDhF Ⅷ gene integration. The results showed that: The successful construction of the lentiviral expression vector pXZ208  BDDhF Ⅷ, the gene transduction has reached 59.57% transfection efficiency. RT  PCR method can detect BDDhF Ⅷ transcription of mRNA. 24,48,72 hours after infection, the cell supernatant was detected in F Ⅷ activity (F Ⅷ: C) were 12%, 43%, 87%. PCR amplified a specific fragment of 534 bp. Conclusion: The successful construction of the lentiviral vector pXZ208  BDDhF Ⅷ, in vitro infection of 293T cells and can be effectively activated expression of the F Ⅷ, tips can be applied to gene therapy for hemophilia A. Keywords: lentivirus Expression of B Domain  Deleted Human Coagulant Factor Ⅷ Gene in 293T Cells Mediated by Lentiviral Vector In Vitro Abstract This study was aimed to construct a lentiviral vector carryin