Construction And Identification Of Eukaryotic Expression Vector And Tet-on Adjustable Recombinant Adenovirus Vector Containing Human NT3 And BDNF Genes

Neurotrophins family is a protein family which has close relationship with nervous system. Human neurotrophin-3(NT3 )gene has 774 bp ,which codes 257 amino acid residues. Human brain-derived neurotrophic factor (BDNF) gene has 744 bp ,which codes 247 amino acid residues.Studies have showed that NT3 and BDNF are very important agents in regenerative micro-environment which can promote the regeneration of peripheral nerve by exerting effect on neuron, injuried local position and target organs.In this study, we have constructed eukaryotic expression vector and Tet-on adjustable recombinant adenovirus vector containing human NT3 and BDNF genes respectively.The procedures and results are as follow:The first part: Construction and identification of eukaryotic expression vector containing human NT3 and BDNF genesThe eukaryotic expression vector marked by enhanced green fluorescent protein which containing human NT3 and BDNF genes were constructed , named as pEGFP-N2-NT3 and pEGFP-N2-BDNF respectively. HepG2 cells were transfected by them throughliposomes, and the expression was detected with green fluorescent and Western Blot.1 .Construction and identification of eukaryotic expression vector pEGFP-N2-NT3 and pEGFP-N2-BDNFWe designed primers for NT3 and BDNF genes which have get rid of their termination codon and give EcoR I and BamHI sites at their both tips. After being amplificated by PCR, the fragments were cloned into pMD-18T vector for sequencing. Then the fragments were cloned into eukaryotic expression vector pEGFP-N2, and were identificated again by enzyme digestion with EcoR I and BamHI.The results showed that eukaryotic expression vector marked by enhanced green fluorescent protein which containing human NT3 and BDNF genes have been constructed successfully.2.Recombinant plasmid pEGFP-N2-NT3 and pEGFP-N2-BDNF transfected HepG2 cellsHepG2 cells were transfected by eukaryotic expression vector pEGFP-N2-NT3 and pEGFP-N2-BDNF through liposomes, and the expression of NT3 and BDNF 36 hours after transfection was detected by green fluorescent and Western Blot.The second part: Construction and identification of Tet-on adjustable recombinant adenovirus vector containing human NT3 and BDNF genesIn order to get highly effective adjustable expression of NT3 and BDNF genes, recombinant adenovirus vector was constructed with Adeno-X Tet-On expression system from BD Bioscience containing a Tet sensitive component for adjusting the expression of taget gene effectively.l.Subcloning of taget genes and Construction and identification of Tet-on adjustable recombinant adenovirus vectorFull length NT3 and BDNF cDNAs were subcloned into pIND vector, followed by being cloned into pTRE-shuttle2 vector. The NT3 and BDNF gene fragments resulted from thepTRE-shuttle2-NT3 and pTRE-shuttle2-BDNF were digested with I-Ceu I and PI-Sce I, and linked to the linear adeno-X virus DNA. The recombinant adenovirus vectors were confirmed by PCR and restriction enzyme digestion analysis.The PCR identification showed that a given band with 3l2bp, and predictive fragments proved by restriction enzyme digestion analysis were exhibited.2. Packaging of virus and the titer determining with end-point dilution assayAfter constructing of Tet inducible recombinant adenovirus vector of Human NT3 and BDNF genes successfully, we packaged them in HEK293 cells,and determined titer by end-point dilution assay.The result showed the titer were 1x108 pfu/ml(Adeno-NT3) and 0.63X108pfu/ml (Adeno-BDNF). All the above results indicated that human NT3 and BDNF genes had been connected with pAdeno-X vectors correctly, and could be used to introduce the target genes into Schwann cells in vitro or in vivo.