Preparation Of PEDF-Fc Fusion Protein And Its Effects On Proliferation And Apoptosis Of Lung Adenocarcinoma A549 Cells

Purpose To construct an eukaryotic expression plasmid pc DNA3.4-PEDF-Fc,the PEDF-Fc fusion protein was expressed and purified in 293 F cells and to obtain high expression,high purity,biologically active PEDF-Fc fusion protein.Then the study was designed to investigate the effect of PEDF-Fc fusion protein alone or in combination with cisplatin on the proliferation and apoptosis of lung adenocarcinoma A549 cells in vitro.Methods The PEDF cDNA was amplified by PCR and the amplified fragment of PEDF c DNA was fused with plasmid pc DNA3.4-Fc encoding the human immunoglobulin Ig G1 Fc region.The recombinant plasmid pc DNA3.4-PEDF-Fc was constructed.The recombinant plasmid was transfected into HEK293 F cells by lipofectamine.The supernatant was eluted and the affinity medium Mabselect Su Re was used to purify the Fc fusion protein,and the ultrafiltration centrifuge tube was used to concentrate the protein.The concentration of fusion protein was determined by BCA method.SDS-PAGE and Western Blot were used to detect and identify the expressed protein.The inhibitory effects of different concentrations of PEDF-Fc(2.5,5,10,20,40?g/m L)fusion protein on the proliferation of HUVECs and A549 cell were observed by MTT assay after 48 hours(P <0.05).MTT assay was used to detect the inhibitory effect of different concentrations of cisplatin(0.625,1.25,2.5,5,10?g/m L)combined with PEDF-Fc(10 ?g/m L)on proliferation of lung adenocarcinoma A549 cells.The effect of different concentrations of PEDF-Fc(10,40?g/m L)on the apoptosis of HUVECs and the effects of different concentrations of PEDF-Fc fusion protein(10,40?g/m L)alone or in combination with cisplatin(2?g/m L)on the apoptosis of A549 cell was detected by flow cytometry.Results Double digests and gene sequencing revealed successful construction of the recombinant pc DNA3.4-PEDF-Fc plasmid and the concentration of PEDF-Fc fusion protein was about 3.65mg/m L tested by BCA.The expression level of PEDF-Fc fusion protein was about 90 mg/L;SDS-PAGE and Western Blot confirmed that PEDF-Fc fusion protein with high purity.The molecular weight of the recombinant PEDF-Fc fusion protein was about 75KDa;MTT results showed that different concentrations of PEDF-Fc fusion protein(2.5,5,10,20,40?g/m L)had proliferative inhibition effect on HUVECs in a certain concentration-dependent manner(P<0.05).When the concentration of PEDF-Fc fusion protein was ?5?g/m L,PEDF-Fc fusion protein could inhibit the proliferation of A549 cells in a dose-dependent manner(P<0.05).Flow cytometry showed that the apoptotic rate of HUVECs was significantly higher in the PEDF-Fc intervention group(10,40?g/m L)than in the control group(P<0.05),and the apoptotic rate in the high concentration intervention group was significantly higher than that in the low concentration intervention group(P <0.05).Compared with PEDF-Fc or cisplatin alone group,PEDF-Fc combined with cisplatin had stronger apoptosis-promoting effect on A549 cells(P <0.05).Conclusions The PEDF-Fc fusion protein was successfully constructed and successfully obtained.The PEDF-Fc fusion protein with high purity and high activity was successfully obtained,which laid a foundation for the study of PEDF protein in the treatment of lung cancer.The results showed that the proliferation inhibition and apoptosis promotion effects of PEDF-Fc fusion protein and low dose cisplatin on lung adenocarcinoma A549 cells was stronger than that of PEDF-Fc fusion protein or cisplatin alone,suggesting that the potential value of PEDF-Fc fusion protein and cisplatin in the treatment of lung cancer is worthy of further study.