title: Expression, purification, and immunogenic characterization of Epstein-Barr virus recombinant EBNA1 protein in Pichia pastoris.
abstract: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390-641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4+ and CD8+ T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China.
authors.forename: Man
authors.initials: M
authors.lastname: Wang
authors.forename: Shuai
authors.initials: S
authors.lastname: Jiang
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Liu
authors.forename: Yefu
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D016207:Cytokines / Q000276:immunology; D020031:Epstein-Barr Virus Infections / Q000276:immunology / Q000821:virology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D005260:Female; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018417:Th1 Cells / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D016207:Cytokines; D019309:Epstein-Barr Virus Nuclear Antigens; D011994:Recombinant Proteins; C417029:EBV-encoded nuclear antigen 1