title: Expression of E2 gene of bovine viral diarrhea virus in Pichia pastoris: a candidate antigen for indirect Dot ELISA.
abstract: The E2 gene containing the EcoR I and Not I sites of bovine viral diarrhea virus (BVDV) was amplified from the plasmid pMD-18T-E2 of the HB-bd isolated, and inserted into Pichia pastoris (P. pastoris) expression vector pPIC9K, and transfected into Escherichia coli DH5α. The recombinant plasmid pPIC9K-E2 was digested by the SalI restriction enzyme and transformed into the P. pastoris strain GS115 by electroporation. High copy integrative transformants were obtained by G418 screening and induced for expression with methanol. The expressed products in the culture medium were identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the Western blotting and the antibody test for immunity. An indirect Dot-ELISA for the detection of antibody against BVDV was established by the recombinant E2 protein as the coating antigen. The reaction conditions of the indirect Dot-ELISA were optimized. The coating concentration of the E2 recombinant protein antigen, the dilution of serum sample, the optimal concentration of HRP labeled antibody, the optimal blocking reagent and blocking time were studied. 100 sera samples from cows in the field were tested for the antibody against BVDV by the Dot-ELISA and the IDEXX HerdChek BVDV antibody ELISA kit simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the expressed products in the culture medium resulted in single band of 44kDa by SDS-PAGE and Western blotting. The results of the immunogenicity assay indicated that the protein E2 expressed in P. pastoris could induce the experimental animals of the rabbit to produce BVDV specific antibodies. The results of the indirect Dot-ELISA showed that the optimal coating concentration of the E2 recombinant protein was 2.0μg/mL, the bovine serum dilution was 1:100, the optimal concentration of HRP-labeled rabbit anti-bovine antibody IgG was 1:500, and the optimal blocking reagent was 3% glutin-TBS and blocking for 45min. The indirect Dot-ELISA showed 96.7%, 92.5% and 95% in the terms of specificity, sensitivity and accuracy compared to the IDEXX ELISA test kit. The indirect Dot-ELISA using the E2 recombinant protein can be used for the detection of antibody against the BVDV and could be considered in the surveillance programs.
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Yuelan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Tianyi
authors.initials: T
authors.lastname: Ma
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Xingyu
authors.initials: X
authors.lastname: Ju
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Cao
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
authors.forename: Yongzhan
authors.initials: Y
authors.lastname: Bao
authors.affiliation: College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China. Electronic address: qjhqqq@126.com.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Qin
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis*; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D018182:Pestivirus Infections / Q000175:diagnosis / Q000662:veterinary*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins
keywords: Bovine viral diarrhea virus; E2 gene; Immunogenicity; Indirect Dot-ELISA