title: Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique.
abstract: Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.
authors.affiliation: Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102, Taiwan.
authors.forename: Peng-Yeh
authors.initials: PY
authors.lastname: Lai
authors.affiliation: Department of Chemical and Biochemical Engineering and Institute of Chemical and Biochemical Engineering, Kao Yuan University, Luzhu District, Kaohsiung City 82151, Taiwan.
authors.forename: Chia-Tse
authors.initials: CT
authors.lastname: Hsu
authors.affiliation: Department of Microbiology, Immunology and Biopharmaceuticals, National Chiayi University, Chiayi City 60004, Taiwan.
authors.forename: Shao-Hung
authors.initials: SH
authors.lastname: Wang
authors.affiliation: Department of Biotechnology, Kaohsiung Medical University, Kaohsiung City 80708, Taiwan.
authors.forename: Jin-Ching
authors.initials: JC
authors.lastname: Lee
authors.affiliation: Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102, Taiwan.
authors.forename: Min-Jen
authors.initials: MJ
authors.lastname: Tseng
authors.affiliation: Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei City 11031, Taiwan.|Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102, Taiwan.
authors.forename: Jaulang
authors.initials: J
authors.lastname: Hwang
authors.affiliation: Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102, Taiwan.
authors.forename: Wen-Tsai
authors.initials: WT
authors.lastname: Ji
authors.affiliation: Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, College of Science, National Chung Cheng University, Min-Hsiung, Chia-Yi 62102, Taiwan.
authors.forename: Hau-Ren
authors.initials: HR
authors.lastname: Chen
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000302:isolation & purification*; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification*; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins