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Enhancing expression of the pseudorabies virus glycoprotein E in yeast and its application in an indirect sandwich
1Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
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.
