Construction of human embryonic kidney cells exhibiting human preproenkephalin gene expression

Abstract

Objective To construct human embryonic kidney cells (HEK293) modified with human preproenkephalin (hPPE) gene.Methods hPPE gene fragments were obtained from recombinant plasmid pcDNA3.1( + )/hPPE by using restriction endonuelease Hind Ⅲ and Not Ⅰ.Homologous recombination of lentivirus and hPPE gene was produced by using recombinant DNA technology.HEK293 cells were then transfected with the recombinant lentivirus vectors.The expression of hPPE gene in HEK293 cells was detected by Western blot.Results The results of DNA sequencing indicated that the positive clone of recombinant lentivirus was completely consistent with sequencing of hPPE in Genebank.The titer of the concentrated virus was 2.07 × 108 TU/ml.GFP fluorescence was not seen in HEK293 cells transfected with the lentiviral vector under fluorescence microscope.A strong fluorescence was seen in HEK293 cells transfected with Ubc-GFP-L.V.empty viral vector.Positive expression of hPPE was demonstrated in HEK293 cells transfected with lentiviral vector by Western blot.Conclusion HEK293 cells modified with hPPE gene were successfully constructed and the target gene hPPE was stably expressed in HEK293 cells. Key words: Enkephalins; Cell line; Organisms,genetically modified; G ene therapy