title: In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.
abstract: The canine Parvovirus 2, non-structural 1 (NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1 (rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS-PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 x His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.
authors.affiliation: Molecular Biology Laboratory, Department of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243 122, India.
authors.forename: G Ravi
authors.initials: GR
authors.lastname: Kumar
authors.forename: Shikha
authors.initials: S
authors.lastname: Saxena
authors.forename: Lovleen
authors.initials: L
authors.lastname: Saxena
authors.forename: Uttara
authors.initials: U
authors.lastname: Chaturvedi
authors.forename: Lakshman
authors.initials: L
authors.lastname: Santra
authors.forename: Rajiv
authors.initials: R
authors.lastname: Kumar
authors.forename: Aditya Prasad
authors.initials: AP
authors.lastname: Sahoo
authors.lastname: Rajmani
authors.forename: Aswani
authors.initials: A
authors.lastname: Kumar
authors.forename: G S
authors.initials: GS
authors.lastname: Desai
authors.forename: Sudesh
authors.initials: S
authors.lastname: Kumar
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Tiwari
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D004285:Dogs; D004926:Escherichia coli; D015870:Gene Expression / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera; D066298:In Vitro Techniques; D017993:Parvovirus, Canine / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D017361:Viral Nonstructural Proteins / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000941:Antigens; D007106:Immune Sera; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins