Construction and expression of recombinant plasmid pcDNA3/NT-3

Objective To construct the eukaryotic expression recombinant plasmid pcDNA3/NT-3 and investigate its expression in L929 cells.Methods The gene of rat neurotrophin 3(NT-3)was amplified by RT-PCR from rat brain tissue.By gene recombination technique,rat NT-3 coding sequence was inserted into eukaryotic expression vector pcDNA3.The recombinant plasmid was verified with restriction enzyme digestion and DNA sequencing.L929 cells were transfected with the recombinant vector using Lipofectamine 2000 transfection reagent.The expression of NT-3 was analyzed by immunocytochemistery as well as western blot.Results The RT-PCR product is 822bp specific segment.By restriction enzyme digestion,the recombinant plasmid was digested into 822bp and 5.2kb fragments.The DNA sequence of the 822bp fragment was identical with rat NT-3 cDNA in GeneBank.The immunocytochemistery and western blot showed the NT-3 was expressed successfully in L929 cells.Conclusions The recombinant plasmid pcDNA3/NT-3 was constructed successfully,which will provide the foundation for the further research.