title: Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle.
abstract: The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.
authors.affiliation: a National Institute of High Security Animal Diseases, Indian Council of Agricultural Research , Bhopal , Madhya Pradesh , India.
authors.forename: Sthita Pragnya
authors.initials: SP
authors.lastname: Behera
authors.forename: Niranjan
authors.initials: N
authors.lastname: Mishra
authors.forename: Ram Kumar
authors.initials: RK
authors.lastname: Nema
authors.forename: Pooja Dubey
authors.initials: PD
authors.lastname: Pandey
authors.forename: Semmannan
authors.initials: S
authors.lastname: Kalaiyarasu
authors.forename: Katherukamem
authors.initials: K
authors.lastname: Rajukumar
authors.forename: Anil
authors.initials: A
authors.lastname: Prakash
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000097:blood* / Q000235:genetics / Q000276:immunology; D002417:Cattle; D001908:Diarrhea Viruses, Bovine Viral; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C076551:gp53, bovine viral diarrhea virus
keywords: E2 glycoprotein; ELISA; Pichia pastoris; bovine viral diarrhea virus; virus neutralization test; yeast