Enhancing expression of the classical swine fever virus glycoprotein E2 in yeast and its application to a blocking ELISA.
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.
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Chih-Yuan
CY
Cheng
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Ching-Wei
CW
Wu
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Guang-Jan
GJ
Lin
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Wei-Cheng
WC
Lee
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.
Maw-Sheng
MS
Chien
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.
Chienjin
C
Huang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology*; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000276:immunology*; D003062:Codon; D004279:DNA, Viral / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000145:classification / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013552:Swine; D022223:Vaccines, Subunit / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D003062:Codon; D004279:DNA, Viral; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; C087086:glycoprotein E2, classical swine fever virus
Blocking ELISA; Classical swine fever virus; Codon optimization; Monoclonal antibody; Subunit marker vaccine