Construction, expression, and bio-activity assay of an anti-IL-1βscfv and TNFR1 fusion protein
10.3760/cma.j.issn.0254-5101.2012.10.004
- VernacularTitle:抗IL-1βscfv抗体与TNF-α可溶性受体融合蛋白的构建及其生物学活性分析
- Author:
Fangming KAN
;
Guiping REN
;
Mo GUO
;
Yang HAN
;
Jianying QI
;
Yu ZHANG
;
Yakun ZHANG
;
Deshan LI
- Publication Type:Journal Article
- Keywords:
IL-1β;
TNF-α;
sTNFR1;
Rheumatoid arthritis
- From:
Chinese Journal of Microbiology and Immunology
2012;(10):855-860
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express the anti-IL-1βscfv and soluble TNF receptor 1 (sTNFR1),and analyze their bio-activities.Methods sTNFR1 was obtained by RT-PCR from the total RNA of HeLa cells,and fused with IL-1βscfv by the hinge fragment of IgG molecule.The fusion gene IL-1scfv:TNFR1 was cloned into the expression vector pET27b(+).The fusion protein was expressed and purified from inclusion bodies.Results The ELISA analysis showed that the fusion protein could bind hIL-1β and hTNF-α respectively in a dose-dependent manner,indicating that scfv and sTNFR in the fusion protein can form the correct spatial configuration.The dolt-blot analysis showed that the fusion protein could concurrently bind with hIL-1β and hTNF-α,indicating that the combination of the two parts of the fusion protein does not influence each other for binding to their target molecules.The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells,indicating that the fusion protein has the ability to neutralize hTNF-α and hIL-1β.Conclusion A bispecific fusion protein IL-1scfv:TNFR1 was successfully constructed.The fusion protein has the ability to inhibit the biological activity of hTNF-α and hIL-1β,and provides a drug candidate for the treatment of rheumatoid arthritis.
