Construction Of The Enhanced Green Fluorescent Protein Eukaryotic Expression Vector Of Human Neurotrophin-3 Gene And Its Expression In Eukaryotic Cells

Objective Neurotrophin-3 ( NT-3 ) is beyond to neurotrophins which are a family of growth factors. NT-3 play an important rols in survival and growth of neurons. To construct the enhanced green fluorescent protein eukaryotic expression vector of Human Neurotrophin-3 gene ( hNT-3 ) .To obtain mutation of Human Neurotrophin 3 gene by Error Prone PCR ( EPPCR ) technology which can introduce random mutations in gene fragment.To determine whether the expression of enhanced green fluorencent protein would change the biological characters of C6 cells in vitro.Methods Genome was extracted from human peripheral blood. Human Neurotrophin 3 gene fragments was amplified by PCR. PCR product and pEGFP-N1 were double digested by EcoR I and BamH I. Human Neurotrophin 3 gene was cloned into pEGFP-N1 to construct recombinat plasmid pEGFP-NT3. pEGFP-NT3 was transinfected into mesenchymal stramatic cells of SD rat and 293T cells.Human Neurotrophin 3 gene was mutated by EPPCR. The mutated Human Neurotrophin 3 gene was ligated with pGEM^?-T Easy Vector.C6 cells were transfected with pEGFP-N1 by lipofectamine Plus. Stable EGFP-expressing cells were isolated with G418. Proliferation, activity and cell cycle of C6-EGFP were evaluated through cell-counting, MTT and Flowcytometry.Results A fragment about 800 bp was obtained through PCR and proved to be...