ABSTRACT
Human fibroblast growth factor 8b (FGF8b) was expressed based on a baculovirus expression vector system (BEVS) and identified as having a protective effect on Parkinson's disease. Immunoblotting demonstrated that rhFGF8b proteins were recognized by a human anti-FGF8b antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield and protein quality. Our results indicated that the rhFGF8b was first detectable at 36 h postinfection and reached a maximum at 60 h. A multiplicity of infection (MOI) of 8 pfu/mL was suitable for harvest. The target protein was purified by heparin-affinity chromatography. In vitro methylthiazol tetrazolium (MTT) assays demonstrated that the purified rhFGF8b could significantly stimulate proliferation of NIH3T3 cells. Furthermore, to elucidate the effect of rhFGF8b on Parkinson's disease, we used FGF8b pretreatment on a cell model of Parkinson's disease. The results indicated that rhFGF8b prevented necrosis and apoptosis of 1-METHYL-4-phenyl pyridine (MPP(+)) treated PC12 cells. Moreover, the effect of FGF8b on messenger RNA (mRNA) levels of apoptosis and ERS genes was investigated to clarify the molecular mechanisms of FGF8b. The results suggest that FGF8b exerts neuroprotective effects by alleviating endoplasmic reticulum (ER) stress during PD. These results suggest that FGF8b may be a promising candidate therapeutic drug for neurodegenerative diseases related to ER stress.
Subject(s)
Fibroblast Growth Factor 8/genetics , Fibroblast Growth Factor 8/pharmacology , Neuroprotective Agents/pharmacology , Animals , Apoptosis/drug effects , Baculoviridae/genetics , Cell Proliferation/drug effects , Cell Survival/drug effects , Chromatography, Affinity , Endoplasmic Reticulum Stress/drug effects , Fibroblast Growth Factor 8/biosynthesis , Fibroblast Growth Factor 8/isolation & purification , Humans , Mice , NIH 3T3 Cells , Neuroprotective Agents/isolation & purification , PC12 Cells , Parkinson Disease/drug therapy , Parkinson Disease/physiopathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Tetrazolium Salts , ThiazolesABSTRACT
cDNA of human fibroblast growth factor 8 isoform b (FGF8b) was cloned for the first time into a plant expression vector with or without endoplasmic reticulum retention signal (KDEL) and was transiently expressed as His tagged fusion protein in Nicotiana tabacum leaves through Agrobacterium mediated gene transfer by vacuum infiltration method. Expression of FGF8b was confirmed by ELISA and Western blot using anti-FGF8b antibody and the expression level was measured as 4.1% of total soluble protein of tobacco leaves. The expressed recombinant protein was purified by Ni-NTA affinity chromatography and its molecular weight was determined by MALDI-TOF-MS. Schiff's test, Concanavalin A (Con A) immunoblot and enzymatic deglycosylation indicated that the high molecular mass was due to glycosylation of the FGF8b expressed in plant cells. Measurement of its biological activity in NIH3T3 cells by thymidine incorporation and MTT assay showed induction of cell proliferation. These results indicate that biologically active recombinant FGF8b could be expressed in tobacco plants.
