Establishment of a high efficient human coagulation factor VIII eukaryotic expression system using lentiviral vector / 中华血液学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To establish a high efficient human coagulation factor VIII (FVIII) eukaryotic stable expression system using lentiviral vector, and determine its biosafety.</p><p><b>METHODS</b>Lentiviral transfer plasmid carrying human B-domain-deleted FVIII(BDDhFVIII)-IRES-GFP(BDDhFVIII/pXZ9)or IRES-GFP(pXZ9) was constructed. Lentivirus particles were produced by transiently co-transfected 3-plasmids into 293FT cells and further concentrated via ultracentrifugation. CHO cells were infected, 72h later, the FVIII antigen (FVIIIAg) concentration in the medium was examined by ELISA, the activity was detected via one stage coagulation,and the transcription of FVIII in the infected CHO cells was determined by RT-PCR.Virus infection ability in the medium and the gag gene in CHO cells were determined to evaluate the model's biosafety.</p><p><b>RESULTS</b>Lentiviral transfer plasmid BDDhFVIII-IRES-GFP(BDDhFVIII/pXZ9)carrying human B-domain-deleted FVIII or IRES-GFP (pXZ9) was successfully constructed, and high titer lentiviruses has been prepared. The lentivirus could infect CHO cells efficiently, after an additional 72 h, the FVIIIAg concentration had up to (1724.9±283.7) mU/ml, the FVIIIC level increased to (10.58±1.55)%, and transcripts of BDDhFVIII mRNA could be measured by RT-PCR. Neither the gag gene nor the virus in the supernatant was detected.</p><p><b>CONCLUSION</b>Lentivirus-mediated human coagulation factor VIII could be expressed efficiently in CHO cells. The system couldn't produce offspring virus, proving a good biosafety.</p>