Construction and identification of CCDC67 gene lentiviral vector / 中华内分泌外科杂志

ABSTRACT

Objective To construct a]entiviral expression vector carrying CCDC67 and to obtain CCDC67 efficient and stable expression in mammalian cells.Methods The DNA fragment of CCDC67 coding sequence was amplified by PCR with designed primer from the cDNA library including CCDC67 gene,and then subcloned into GV208 vector with in-fusion technique to generate the lentiviral expression vector,GV208-CCDC67.The positive clones were screened by PCR and the correct CCDC67 was confirmed by sequencing.Recombinant lentiviruses were produced in 293T cells after the cotransfection of GV208-CCDC67,and packaging plasmids of pHelper 1.0 and pHelper 2.0 Green fluorescent protein (GFP)expression in 293T cells was observed to evaluate gene delivery efficiency.CCDC67 protein expression in 293T cells was detected by Western blot.Resuits The lentiviral expression vector carrying CCDC67 was successfully constructed and CCDC67 protein expression was detected in 293T cells.Conclusion The recombinant lentivirus GV208-GFP-CCDC67 is successfully constructed and it lays a foundation for further molecular function study of CCDC67.