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Enhancing expression of the pseudorabies virus glycoprotein E in yeast and its application in an indirect sandwich ELISA
Author(s) -
Wu C.Y.,
Wu C.W.,
Liao C.M.,
Chien M.S.,
Huang C.
Publication year - 2017
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13531
Subject(s) - pseudorabies , pichia pastoris , antigenicity , glycoprotein , microbiology and biotechnology , antigen , recombinant dna , virology , biology , glycosylation , virus , antibody , yeast , gene , chemistry , biochemistry , genetics
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) ( gEN 31–270) was codon optimized and expressed constitutively and secreted using a Pichia pastoris expression system. Yeast‐expressed gEN 31–270 (yg EN 31–270) was harvested from the culture supernatant, and yg EN 31–270 was shown to exhibit N‐linked glycosylation. An indirect sandwich enzyme‐linked immunosorbent assay ( ELISA ) was developed using yg EN 31–270 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. yg EN 31–270 was secreted and N‐glycosylated. The yg EN 31–270‐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 yg EN 31–270‐based indirect sandwich ELISA may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.