Study On The Construction Of Nerve Growth Factor Beta Subunit Gene Lentiviral Vector

BackgroundDiabetic neurogenic bladder,characterized by bladder dysfunction, is one of common refractory chronic complications of diabetes mellitus.Recently some researchers argue that diabetic neurogenic bladder is probably related to the deficiency of neurotrophic factors in focus. Neurotrophic factors supplement locally may contribute to improving the function of diabetic bladder. Nerve growth factor (NGF) composed of three subunits is one of the most important neurotrophic factors. Beta subunit (beta-NGF) is the only biologically active subunit,which is indispensible to maintain growth development and normal function of sympathetic nerve and sensory nerve. Studies indicate that transfection and expression of NGF gene through suitable vector locally can receive satisfactory therapeutic results. Lentiviral vector can infect separated and unseparated cells,transfer comparatively large gene fragment or introduce two different genes into one cell,express foreign gene for a long time,rarely induce the host immune response,or infect the same cell repeatedly,therefore it is an ideal vector for gene therapy.Thus,we plan to construct a recombinant lentiviral vector which contains human nerve growth factor beta subunit in this study to lay a foundation for gene therapy in vivo in the future.Objective To construct a lentiviral vector carrying human nerve growth factor beta subunit gene, which may lay a foundation for beta-NGF gene modified rat bone marrow-derived mesenchymal stem cells (BMSCs) and their transplantation therapy for diabetic neurogenic bladder.Methods Beta-NGF gene was amplified from plasmid pGC-E1-NGFB by PCR and subcloned into the lentiviral vector pGC-FU, to generate the lentiviral expression vector, pGC-FU-beta-NGF. The sequence of beta-NGF was confirmed by restriction enzyme digestion and sequencing. Recombinant lentiviruses GC-FU-beta-NGF were produced by packaging cell line 293T cells following the co-transfection of pGC-FU- beta-NGF, with the packaging plasmids pHelper1.0 and envelop plasmids pHelper2.0. The titer of recombinant lentiviruses was measured by real-time quantitative PCR.The recombinant lentiviruses which carrying beta-NGF gene were then used to infect 293T cells , beta-NGF expression in 293T cells was dectected by reverse transcription polymerase chain reaction and Western blotting analysis.Rusults The result of sequencing showed the sequence of the cloned beta-NGF gene was consistent with that was reported in the GeneBank. Plasmid pGC-FU- beta-NGF carried the correct beta-NGF gene. The recombinant lentiviruses GC-FU- beta-NGF which carry beta-NGF gene could be produced by co-transfection pGC-FU- beta-NGF to 293T cells with pHelper1.0 and pHelper2.0. Recombinant lentiviruses measured by real-time quantitative PCR in high titer were obtained( 2.43E+9 TU/ml).The recombinant lentiviruses GC-FU-beta-NGF could infect and deliver beta-NGF gene to 293T cells efficiently, beta-NGF protein expression was confirmed by reverse transcription polymerase chain reaction and Western blotting.Conclusions The recombinant lentiviruses GC-FU-NGF can be produced successfully in high titer with correct expression.