title: Production of Influenza Virus HA1 Harboring Native-Like Epitopes by Pichia pastoris.
abstract: The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Qingshan
authors.initials: Q
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Kunyu
authors.initials: K
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Fangping
authors.initials: F
authors.lastname: He
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Zhenqin
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.|National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China. shaowei@xmu.edu.cn.|National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China. shaowei@xmu.edu.cn.
authors.forename: Shaowei
authors.initials: S
authors.lastname: Li
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China. qinjian_zhao@xmu.edu.cn.
authors.forename: Qinjian
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.|National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen, Fujian, 361002, People's Republic of China.
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics; D000939:Epitopes / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006385:Hemagglutination Inhibition Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007252:Influenza Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007251:Influenza, Human / Q000235:genetics / Q000276:immunology* / Q000517:prevention & control; D029524:Betainfluenzavirus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D051379:Mice; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000914:Antibodies, Viral; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C493831:hemagglutinin HA1, influenza B virus
keywords: Fed-batch fermentation; Hemagglutinin; Monoclonal antibody; Native epitopes; Pichia pastoris