Expression,purification,characterization and subcellular localization of the goose parvovirus rep1 protein

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. © 2013 bentham science publishers.