Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
J Biotechnol. 2014 Mar 20;174:1-6. doi: 10.1016/j.jbiotec.2014.01.007. Epub 2014 Jan 24.
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.
经典猪瘟病毒（CSFV）感染是一种严重的猪病，常导致巨大的经济损失。毕赤酵母表达的 CSFV 糖蛋白 E2（yE2）已被证明能诱导针对该病毒的保护性免疫反应。为了提高 yE2 的表达水平，将 E2 基因的第一个密码子 Arg（CGG）优化为最常用的密码子 AGA。在密码子优化的菌株（N342）中，E2 蛋白的产量显著增加。还构建了三个截短的 E2 亚基，分别编码 N 端 330（N330）、301（N301）和 190（N190）残基。通过对猪进行免疫接种，证实了每个重组 E2 亚基的免疫原性，所有免疫组在加强免疫后均产生了高中和抗体滴度，并持续了很长时间。此外，还产生了一种针对 yE2 的单克隆抗体（MAb）1B6，并显示出可识别 CSFV 感染细胞。用 N330 作为包被抗原，用辣根过氧化物酶标记的 MAb 1B6 进行基于阻断酶联免疫吸附试验（ELISA），对一系列猪血清进行了检测，该检测具有很高的灵敏度和特异性。重组 yE2 亚基可能为 CSFV 提供潜在的亚单位疫苗候选物和有用的诊断试剂，具有易于操作和低成本的特点。
