Preparation and Identification of a Novel FGF20 Monoclonal Antibody

doi:10.13560/j.cnki.biotech.bull.1985.2020-1525

Abstract

Abstract:

Prokaryotic expression vector for expression recombinant fibroblast growth factor 20（FGF20）was constructed,and monoclonal antibody against FGF20 was prepared,which may lay foundation for developing novel medicines. The prokaryotic expression vector pET24a-SUMO-hFGF20 was constructed and transformed into Escherichia coli BL21（DE3）for induction and expression. The fusion protein was purified by Ni NTA affinity chromatography column,digested with SUMO enzyme,and purified again by Ni NTA affinity chromatography column. The purity of the protein was detected by SDS-PAGE. Balb/c mice were immunized with the recombinant FGF20 protein,and the monoclonal antibody was prepared by conventional way,and isolated and purified by protein G column. The purity of the antibody was detected by SDS-PAGE. The titer and subtype of the antibody were detected by ELISA. The affinity of the antibody was determined by Western blot and SPR. The recombinant plasmid pET24a-SUMO-hFGF20 was successfully constructed. The relative molecular weight of the isolated and purified FGF20 protein was 23 kD. A highly positive hybridoma cell line detected by ELISA was obtained. After purification,the titer of the monoclonal antibody was 1:25 600,and the subtype is IgG1. It bound to commercial antigen,and the affinity was 1.07×10 ^-7 M. High-affinity monoclonal antibody against the FGF20 protein was successfully prepared by construction,expression and purification of recombinant protein.

Key words: fibroblast growth factor 20（FGF20）, prokaryotic expression, protein purification, monoclonal antibody

TANG Lu, DONG Li-ping, YIN Mo-li, LIU Lei, DONG Yuan, WANG Hui-yan. Preparation and Identification of a Novel FGF20 Monoclonal Antibody[J]. Biotechnology Bulletin, 2021, 37(10): 179-185.

Figures/Tables 8

Fig.1 Double enzyme digestion of plasmid M1:DL5 000 DNA marker. 1:Plasmid enzyme. 2:Plasmid unenzymed. M2:DL2 000 DNA marker

Fig.2 Electrophoregram analysis of soluble of engineere bacteria (SDS-PAGE) M:Protein marker. 1:Before induced. 2:Precipitation after crushing. 3:Supernatant after crushing

Fig.3 Electrophoregram analysis of purified FGF20 M:Protein marker. 1:Enzymed by SUMO. 2:Unenzymed by SUMO. 3:FGF20

Fig.4 Electrophoregram analysis of purified monoclonal antibody M:Protein marker. 1:Ascites. 2:Purified antibody

Fig.5 Antibody titer assay

Fig.6 Antibody subgroup assay

Fig.7 Western Blot assay 1:Purified antigen. 2:Commercial antigen

Fig.8 Kinetic assay on antibody binding to FGF20 antigen by SPR

References 27

[1]	Imamura T. Physiological functions and underlying mechanisms of fibroblast growth factor(FGF)family members:recent findings and implications for their pharmacological application[J]. Biol Pharm Bull, 2014, 37(7):1081-1089. pmid: 24988999
[2]	Ornitz DM, Itoh N, Fibroblast growth factors[J]. Genome Biology, 2001, 2(3):3005.1-3005.12.
[3]	赵央, 田海山, 李校堃, 等. 成纤维细胞生长因子20的研究进展[J]. 中国生物工程杂志, 2015, 35(8):103-108.
	Zhao Y, Tian HS, Li XK, et al. The research progress of fibroblast growth factor 20[J]. China Biotechnology, 2015, 35(8):103-108.
[4]	Narum DL, Kumar S, Rogers WO, et al. Codon optimization of gene fragments encoding plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice[J]. Infection Immun, 2001, 69(12):7250-7253. doi: 10.1128/IAI.69.12.7250-7253.2001 URL
[5]	Ohmachi S, Watanabe Y, Mikami T, et al. FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain[J]. Biochem Biophys Res Commun, 2000, 277(2):355-360. doi: 10.1006/bbrc.2000.3675 URL
[6]	Kirikoshi H, Sagara N, Saitoh T, et al. Molecular cloning and characterization of human FGF-20 on chromosome 8p21. 3-p22[J]. Biochem Biophys Res Commun, 2000, 274(2):337-343. doi: 10.1006/bbrc.2000.3142 URL
[7]	Hou JZ, Kan MK, McKeehan K, et al. Fibroblast growth factor receptors from liver vary in three structural domains[J]. Science, 1991, 251(4994):665-668. pmid: 1846977
[8]	Jaye M, Schlessinger J, Dionne CA. Fibroblast growth factor receptor tyrosine kinases:molecular analysis and signal transduction[J]. Biochem Biophys Acta, 1992, 1135(2):185-199.
[9]	Takagi Y, Takahashi J, Saiki H, et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a parkinson’s primate model[J]. J Clin Invest, 2005, 115(1):102-109. doi: 10.1172/JCI21137 URL
[10]	Hajihosseini MK, Heath JK. Expression patterns of fibroblast growth factors-18 and -20 in mouse embryos is suggestive of novel roles in calvarial and limb development[J]. Mech Dev, 2002, 113(1):79-83. doi: 10.1016/S0925-4773(01)00656-6 URL
[11]	Xu XG, Gong L, Jiang TL, et al. Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20[J]. Biotechnol Lett, 2018, 40(6):1009-1014. doi: 10.1007/s10529-018-2554-z URL
[12]	Yang LM, Huh SH, Ornitz DM. FGF20-expressing, wnt-responsive olfactory epithelial progenitors regulate underlying turbinate growth to optimize surface area[J]. Dev Cell, 2018, 46(5):564-580. doi: S1534-5807(18)30595-1 pmid: 30100263
[13]	Jeffers M, McDonald WF, Chillakuru RA, et al. A novel hunman fibroblast growth factor treats experimental intestinal inflammation[J]. Gastroenterology, 2002, 123(4):1151-1162. doi: 10.1053/gast.2002.36041 URL
[14]	Pan J, Li H, Wang Y, et al. Fibroblast growth factor 20(FGF20)polymorphism is a risk factor for Parkinson’s disease in Chinese population[J]. Parkinsonism Relat Disord, 2012, 18(5):629-631. doi: 10.1016/j.parkreldis.2012.01.017 URL
[15]	Jeffers M, Shimkets R, Prayaga S, et al. Identification of a novel human fibroblast growth factor and characterization of its role in oncogenesis[J]. Cancer Research, 2001, 61(7):313l-3138.
[16]	刘萍, 李云峰, 张捷, 等. FGF20在结直肠癌中的表达及临床意义[J]. 实用癌症杂志, 2009, 24(4):358-359.
	Liu P, Li YF, Zhang Z, et al. The expression of FGF-20 and its clinical significance in the tissues from colorectal cancer[J]. The Practical Journal of Cancer, 2009, 24(4):358-359.
[17]	刘萍, 杨之斌, 李强, 等. 结直肠癌组织中FGF20表达与微血管的相关性[J]. 现代肿瘤医学, 2015, 23(10):1398-1402.
	Liu P, Yang ZB, Li Q, et al. Expression of FGF20 in colorectal cancer and its correlation with microvessel density[J]. Modern Oncology, 2015, 23(10):1398-1402.
[18]	Tabarés-Seisdedos R, Rubenstein JLR. Chromosome 8p as a potential hub for developmental neuropsychiatric disorders:implications for schizophrenia, autism and cancer[J]. Mol Psychiatry, 2009, 14(6):563-589. doi: 10.1038/mp.2009.2 URL
[19]	Baffa R, Santoro R, Bullrich F, et al. Definition and refinement of chromosome 8p regions of loss of heterozygosity in gastric cancer[J]. Clin Cancer Res, 2000, 6(4):1372-1377. pmid: 10778965
[20]	Fitzmaurice C, Abate D, Abbasi N, et al. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 29 cancer groups, 1990 to 2017:a systematic analysis for the global burden of disease study[J]. JAMA Oncol, 2019, 5(12):1749-1768. doi: 10.1001/jamaoncol.2019.2996 pmid: 31560378
[21]	Matsuda Y, Ueda J, Ishiwata T, et al. Fibroblast growth factor receptor 2:expression, roles, and potential as a novel molecular target for colorectal cancer[J]. Patholog Res Int, 2012, 2012(574768):1-8.
[22]	Cao ZX, Zheng RL, Lin HJ, et al. SKLB610:a novel potential inhibitor of vascular endothelial growth factor receptor tyrosine kinases inhibits angiogenesis and tumor growth in vivo[J]. Cell Physiol Biochem, 2011, 27(5):565-574. doi: 10.1159/000329978 URL
[23]	Katoh M, Cancer genomics and genetics of FGFR2[J]. Int J Oncol, 2008, 33(2):233-237.
[24]	Chamorro MN, Schwartz DR, Vonica A, et al. FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development[J]. The EMBO Journal, 2005, 24(1):73-84. doi: 10.1038/sj.emboj.7600460 URL
[25]	Katoh M, Katoh M. FGF signaling network in the gastrointestinal tract[J]. Int J Oncol, 2006, 29(1):163-168
[26]	Katoh M. Networking of WNT, FGF, Notch, BMP, and hedgehog signaling pathways during carcinogenesis[J]. Stem Cell Rev, 2007, 3(1):30-38. doi: 10.1007/s12015-007-0006-6 URL
[27]	姜治国. FGF-20在肝癌中表达的生物学意义及其调控途径初步分析[D]. 上海:第二军医大学, 2005.
	Jiang ZG. Biological Implications of FGF-20 expression in human hepatocellular carcinoma and its regulation pathway analysis[D]. Shanghai:Second Military Medical University, 2005.