title: High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody.
abstract: VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni(2+) affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. coli and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity.
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
authors.forename: Chuanfeng
authors.initials: C
authors.lastname: Li
authors.forename: Zongyan
authors.initials: Z
authors.lastname: Chen
authors.forename: Chunchun
authors.initials: C
authors.lastname: Meng
authors.forename: Lu
authors.initials: L
authors.lastname: Li
authors.forename: Guangqing
authors.initials: G
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000032:analysis; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D004371:Hepatitis Virus, Duck / Q000235:genetics / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins