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J Biotechnol. 2014 Mar 20;174:1-6. doi: 10.1016/j.jbiotec.2014.01.007. Epub 2014 Jan 24.

Enhancing expression of the classical swine fever virus glycoprotein E2 in yeast and its application to a blocking ELISA.

Author information

1
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
2
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
3
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC. Electronic address: mschien@dragon.nchu.edu.tw.
4
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC. Electronic address: cjhuang@dragon.nchu.edu.tw.

Abstract

Classical swine fever virus (CSFV) infection is a severe swine disease, often causing large economic losses. A Pichia pastoris yeast-expressed CSFV glycoprotein E2 (yE2) has been shown to induce a protective immune response against the virus. To improve the expression level of yE2, the first codon of E2 gene, Arg (CGG), which is the least used in P. pastoris, was optimized to the most favorite codon AGA. The yield of E2 protein was remarkably increased in the codon optimized strain (N342). Three truncated E2 subunits encoding the N-terminal 330 (N330), 301 (N301), and 190 (N190) residues, respectively, were also constructed. The immunogenicity of each recombinant E2 subunits was confirmed by immunization of pigs, and all immunized groups demonstrated high neutralizing antibody titers after boost immunization, which lasted for a long period of time. In addition, a monoclonal antibody (MAb), 1B6, specific to yE2, was generated and shown to recognize CSFV-infected cells. A panel of swine sera were tested by peroxidase-conjugated MAb 1B6-based blocking enzyme-linked immunosorbent assay (ELISA) using N330 as coated antigen, and the assay demonstrated high sensitivity and specificity. The recombinant yE2 subunits may provide potential subunit vaccine candidates and useful diagnostic reagents for CSFV with easy manipulation and low cost.

KEYWORDS:

Blocking ELISA; Classical swine fever virus; Codon optimization; Monoclonal antibody; Subunit marker vaccine

PMID:
24468422
DOI:
10.1016/j.jbiotec.2014.01.007
[Indexed for MEDLINE]

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