title: Expression, purification, characterization and subcellular localization of the goose parvovirus rep1 protein.
abstract: The goose parvovirus (GPV) Rep1 protein is both essential for viral replication and a potential target for GPV diagnosis, but its protein characterization and intracellular localization is not clear. We constructed a recombinant plasmid, pET28a/GPV-Rep1, and expressed the Rep1 gene in BL21 (DE3) Escherichia coli. A protein approximately 75 kDa in size was obtained from lysates of E. coli cells expressing the recombinant plasmid. SDS-PAGE analysis showed that after induction with 0.6 mM isopropyl β-D-thiogalactosidase (IPTG) at 30°C for 5 h, the Rep1 protein was highly overexpressed. Two methods used to purify proteins, a salinity-gradient elution and Ni-NTA affinity chromatography, were performed. The amount of Rep1 protein obtained by Ni-NTA affinity chromatography was 41.23 mg, while 119.9 mg of Rep1 protein was obtained by a salinity-gradient elution from a 1 L E. coli BL21 (DE3) culture. An immunogenicity analysis showed that the protein could significantly elicit a specific antibody response in immunized goslings compared to control groups. Antibody titers peaked to 1:5120 (optical density (OD) 450 = 3.9) on day 28 after immunization but had mean titers of 1:10,240 (OD450 = 4.2) in gosling groups immunized with a commercially available GPV-attenuated vaccine strain. Experiments examining subcellular localization showed that the Rep1 protein appeared to associate predominantly with the nuclear membrane, especially during later times of infection. This work provides a basis for biochemical and structural studies on the GPV Rep1 protein.
authors.affiliation: National Engineering Research Center for Poultry, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518 Ziyue Road, Minhang District, Shanghai 200241, China. zychen@shvri.ac.cn
authors.forename: Zongyan
authors.initials: Z
authors.lastname: Chen
authors.forename: Chuanfeng
authors.initials: C
authors.lastname: Li
authors.forename: Gaojing
authors.initials: G
authors.lastname: Peng
authors.forename: Guangqing
authors.initials: G
authors.lastname: Liu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005777:Geese; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010321:Parvoviridae / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins