Enhancing expression of the pseudorabies virus glycoprotein E in yeast and its application in an indirect sandwich ELISA

• Published in: Journal of applied microbiology Vol. 123; no. 3; pp. 594 - 601
• Main Authors: Wu, C.‐Y., Wu, C.‐W., Liao, C.‐M., Chien, M.‐S., Huang, C.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-09-2017
• Subjects: Amino acids; Animals; Antibodies; Antibodies, Viral - analysis; Antibodies, Viral - blood; Antigenicity; Antigens; codon optimization; Codons; Diagnostic systems; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Glycoprotein E; Glycoproteins; Glycosylation; Herpesvirus 1, Suid - genetics; Herpesvirus 1, Suid - immunology; Herpesvirus 1, Suid - isolation & purification; indirect sandwich ELISA; Livestock; Low cost; Optimization; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Pseudorabies - blood; Pseudorabies - diagnosis; Pseudorabies - virology; Pseudorabies virus; Recombinant Proteins - analysis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Sensitivity; Sensitivity and Specificity; Swine; Viral Envelope Proteins - analysis; Viral Envelope Proteins - classification; Viral Envelope Proteins - genetics; Viral Envelope Proteins - immunology; Viruses; Yeast; glycoprotein E; Pseudorabies virus; codon optimization; Pichia pastoris; indirect sandwich ELISA
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.13531

Aims
The purpose of this study was to produce a recombinant pseudorabies virus (PRV) glycoprotein E (gE) protein with the correct antigenicity for use as a low‐cost diagnostic antigen.
Methods and Results
The gene fragment encoding the amino‐terminal immunodominant region of PRV gE (codons 31–270) (gEN31–270) was codon optimized and expressed constitutively and secreted using a Pichia pastoris expression system. Yeast‐expressed gEN31–270 (ygEN31–270) was harvested from the culture supernatant, and ygEN31–270 was shown to exhibit N‐linked glycosylation. An indirect sandwich enzyme‐linked immunosorbent assay (ELISA) was developed using ygEN31–270 as a coating antigen, and the results showed that the assay had high sensitivity and specificity, as well as almost perfect concordance with a commercial gE ELISA kit.
Conclusions
The immunodominant region (amino acids 31–270) of gE was expressed successfully in P. pastoris using a codon optimization strategy. ygEN31–270 was secreted and N‐glycosylated. The ygEN31–270‐based indirect sandwich ELISA showed high sensitivity and specificity to detect gE‐specific antibodies in swine serum samples.
Significance and Impact of the Study
The ygEN31–270‐based indirect sandwich ELISA may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.