title: Production and diagnostic application of a purified, E. coli-expressed, serological-specific chicken anaemia virus antigen VP3.
abstract: The aim of this study was to evaluate the production of chicken anaemia virus VP3 protein in different Escherichia coli strains and to address the diagnostic application of purified E. coli-expressed VP3 protein for the detection of chicken anaemia virus (CAV) infection and the development of an ELISA kit. Three E. coli strains, BL21, BL21 codonplus RP and BL21 pLysS, each harbouring a VP3 protein expressing plasmid, were investigated after induction to produce recombinant VP3 protein. After isopropyl-β-D-thiogalactoside (IPTG) induction, VP3 protein was successfully expressed in all three E. coli strains. The BL21 pLysS strain gave the best performance in terms of protein productivity and growth profile. In addition, the optimal culture temperature and IPTG concentration were found to be 0.25 mM and 20 °C, respectively. Using Ni-NTA-purified VP3 protein as an ELISA coating antigen, the purified VP3 was shown to be highly antigenic and able to discriminate sera from chickens infected with CAV from those that were uninfected during an evaluation of CAV infection serodiagnosis. A VP3-based ELISA demonstrated 100% (6/6 x 100%) specificity and sensitivities of 91.3% (21/23 x 100%) and 82.6% (19/23 x 100%) using cut-off values of the mean plus 2 SD and the mean plus 3 SD, respectively.
authors.affiliation: School of Chinese Medicine Resources, China Medical University, Taichung, Taiwan.
authors.forename: M-S
authors.initials: MS
authors.lastname: Lee
authors.forename: Y-M
authors.initials: YM
authors.lastname: Chou
authors.forename: Y-Y
authors.initials: YY
authors.lastname: Lien
authors.forename: M-K
authors.initials: MK
authors.lastname: Lin
authors.forename: W-T
authors.initials: WT
authors.lastname: Chang
authors.forename: H-Z
authors.initials: HZ
authors.lastname: Lee
authors.forename: M-S
authors.initials: MS
authors.lastname: Lee
authors.forename: G-H
authors.initials: GH
authors.lastname: Lai
authors.forename: H-J
authors.initials: HJ
authors.lastname: Chen
authors.forename: C-H
authors.initials: CH
authors.lastname: Huang
authors.forename: W-H
authors.initials: WH
authors.lastname: Lin
mesh_terms: D000818:Animals; D000956:Antigens, Viral; D036022:Capsid Proteins / Q000276:immunology*; D017727:Chicken anemia virus / Q000276:immunology* / Q000302:isolation & purification; D002645:Chickens / Q000821:virology; D004926:Escherichia coli / Q000821:virology*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; C091717:VP3 protein, Chicken anemia virus