Wu C-Y, Wu C-W, Liao C-M, Chien M-S, Huang C
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
Research Center for Animal Medicine, National Chung Hsing University, Taichung, Taiwan.
J Appl Microbiol. 2017 Sep;123(3):594-601. doi: 10.1111/jam.13531. Epub 2017 Jul 31.
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.
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.
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.
The ygEN31-270-based indirect sandwich ELISA may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.
本研究旨在制备具有正确抗原性的重组伪狂犬病病毒（PRV）糖蛋白E（gE）蛋白，用作低成本诊断抗原。
对编码PRV gE氨基末端免疫优势区（第31 - 270位密码子）（gEN31 - 270）的基因片段进行密码子优化，并使用毕赤酵母表达系统进行组成型表达和分泌表达。从培养上清中收获酵母表达的gEN31 - 270（ygEN31 - 270），结果显示ygEN31 - 270具有N - 糖基化修饰。以ygEN31 - 270作为包被抗原建立间接夹心酶联免疫吸附测定（ELISA），结果表明该测定具有高灵敏度和特异性，与市售gE ELISA试剂盒的检测结果几乎完全一致。
采用密码子优化策略在毕赤酵母中成功表达了gE的免疫优势区（第31 - 270位氨基酸）。ygEN31 - 270实现了分泌表达且具有N - 糖基化修饰。基于ygEN31 - 270的间接夹心ELISA对猪血清样本中gE特异性抗体检测具有高灵敏度和特异性。
基于ygEN31 - 270的间接夹心ELISA可为开发操作简便、成本低廉的诊断试剂盒提供一种替代方法。
