Cloning Of CTGF Gene,Construction Of Eukaryotic Expression Vector And Expression In Human Embryonic Kidney 293 Cells

BackgroundMany angiogenic factors,angiogenic factors and antiangiogenic factors have been involved in the pathogenesis and progression of Diabetic retinopathy(DR).Connective tissue growth factor(CTGF),as regulator of cell matrix,plays an important role in the physiological and pathological processes of cell proliferation,adhesion,migration and angiogenesis,inflammatory response and tissue remodeling.In recent years,the relationship between CTGF and diabetes and its complications has been paid more attention.However,the role and mechanism of CTGF in DR have not been fully elucidated.ObjectiveTo clone human CTGF gene cDNA and to constructrecombinant expression vector pCTGF which was transfectedinto human embryo kidney 293(HEK293)cells.To detect the expression levels of recombinant CTGF mRNA and protein in transfected cells and the effect on cell proliferation,which lay an experimental basis for preparation of CTGF antibody,regulation and function of CTGF expression.MethodsAccording to the gene sequence of human CTGF in the GenBank database,the specific primers were designed.Using the human embryo lung fibroblast HFL-I cell cDNA as a template,the CTGF cDNA was amplified by PCRand was sequenced.The correct CTGF cDNA gene was cloned to the eukaryotic expression vector pEGFP,and the recombinant vector named pCTGF were identified by double enzyme digestionand transfected into HEK293 cells.The effect of expression vector on the growth and proliferation of HEK293 cells was detected by MTT,and the expression levels of mRNA and protein in the transfected cells were detected by real-time fluorescent quantitative PCR(qPCR)and Western Blot.Results1.The cDNA sequence of human CTGF gene was obtained by PCR amplification and sequencing,and the results of double enzyme digestion and sequencing showed that the eukaryotic expression vectorpCTGF containing CTGF cDNA sequence was constructed.2.The results of qPCR and Western Blot experiments showed that CTGF mRNA and protein levels in HEK293 cells transfected with pCTGFsignificantlyincreased.3.Compared with normal HEK293 cells,the proliferation of the pCTGF-transfected cells significantlyincreased(P<0.05).Conclusions1.The cDNA sequence of human CTGF gene was cloned and the eukaryotic expression vector pCTGF was successfully constructed.2.The mRNA and protein expression levels of CTGF gene in HEK293 cells transfected with pCTGF significantlyincreased.3.The increased expression of CTGF gene can promote the proliferation of HEK293 cells with transfection of pCTGF.