ABSTRACT
@#Objective To express recombinant human interferon λ1(rhIFNλ1)by transient transfection in HEK-293F cells and identify it. Methods Two signal peptides[T cell receptor(TCR)and nature signal peptide(NSP)],three vectors[pcDNA3.4,ubiquitous chromatin opening element(UCOE)and PFR]and the target gene rhIFNλ1 were used to construct recombinant plasmids of six signal peptide-vector combinations. Using HEK-293F as host cells,the recombinant plasmids were transfected transiently to express rhIFNλ1 on a shake flask scale. The recombinant plasmid UCOE-Q46-λ with low glycosylation rhIFNλ1 was constructed by using NSP and vector UCOE,and transfected transiently into HEK-293F cells. The expressed product was purified by cation exchange chromatography(HiTrap SP FF),blue gel chromatography(HiTrap Blue HP)and gel filtration chromatography(Sephacryl S-100 HR),which was then analyzed by Western blot and reversed-phase HPLC,and determined for its molecular mass and N-terminal amino acid sequence by mass spectrometry. Results Six recombinant plasmids were constructed correctly as identified by double enzyme digestion and sequencing. With the extension of transfection time,the expression levels of the six expressed products increased gradually,and reached the highest level of10 ～ 20 mg/L at the 6th day of transfection. Double enzyme digestion and sequencing identification proved that the recombinant plasmid UCOE-Q46-λ with low glycosylation rhIFNλ1 was constructed correctly. The recombinant plasmid UCOE-Q46-λwas transfected into HEK-293F cells for 6 d. The purified product of cell culture supernatant showed a relative molecular mass of about 27 800 and a purity of 97. 372%,which showed specific binding to mouse anti-human IL-29/IFNλ1 monoclonal antibody;Two peaks were detected by reversed-phase HPLC,and the peak was showed at 15. 6 min and 20. 0 min respectively;The mass spectrometry molecular mass was about 24 000;The N-terminal five amino acids were G-P-V-P-T.Conclusion The rhIFNλ1 expressed by HEK-293F cells has high purity,which lays a foundation of further study of the protein.
ABSTRACT
@#Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.