Expression and identification of recombinant human interferon λ1 in HEK-293F cells
10.13200/j.cnki.cjb.003959
- VernacularTitle:重组人干扰素λ1在HEK-293F细胞中的表达及鉴定
- Author:
YAN Jiali
- Publication Type:Journal Article
- Keywords:
Recombinant human interferonλ1(rhIFNλ1);
HEK-293F cells;
Signal peptides;
Vector
- From:
Chinese Journal of Biologicals
2023;36(8):911-917
- CountryChina
- Language:Chinese
-
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.
