The Construction Of The Human NGF β Recombinant Adenovirus Vector

Objective The purpose of this study was to construct a recombinant adenovirus vector containing human beta-nerve growth factor gene(h NGF β ) by the homologous recombination in bacteria for investigating the gene therapy of h NGF β gene in chronic pain by the construction of Ad- hNGF β .Methods hNGF β gene was digested from pBLAST44-hNGF β v02, cloned into plasmid pBluescript â…¡ sk(+) and formed plasmid pBluescript â…¡ sk(+)-hNGF β .Then hNGF β gene was digested from plasmid pBluescript â…¡ sk(+)-hNGF β ,inserted into shuttle plasmid pShuttle-CMV The resultant plasmid pShuttle-CMV-hNGF β was linealized with Pme I and transformed into ultracompletent BJ5183 containing pAdeasy-1.Positive clone of homologous recombinant was selected for kanamymic resistance and confirmed by restriction endonuclease analyses.The product of homologous recombination (pAdeasy-1- hNGF β) was transfected into Ad293 cell,in which the recombinant Adenovirus vector was packaged and amplified. The identification was performed by enzyme digestion and PCRResults There were 30% positive recombinant after the linealinzed pShuttle-CMV-hNGF β was transformed into ultracompletent BJ5183 containing pAdEasy-1. The viral titer was 2.24 × 10¹² PFU/L .Enzyme digestion and PCR indicated that the recombinant adenovirus plasmid pAdEasy-1-hNGF β contained hNGF β gene.Conclusion The homologous recombination in bacteria is a convenient and efficient method to prepare recombinant adenovirus plasmid of pAdEasy-1-hNGF β .This offers a good gene transfer vector for the gene therapy in pain management and makes the basis for further research and transgene therapy for chronic pain.