He Haijuan, Qi Baojie, Yang Yongbo, Cui Xiaowen, Chen Tianfeng, Cai Xuehui, An Tongqing, Wang Shujie
National Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150068, China.
Institute of Animal Husbandry, Heilongjiang Academy of Agriculture Sciences, Harbin 150086, China.
Vet Sci. 2023 Aug 5;10(8):506. doi: 10.3390/vetsci10080506.
Serological testing is an important method for the diagnosis of pseudorabies virus (PRV) infection. We aimed to investigate the envelope glycoprotein I (gI) of PRV, a strong immunogen, and its potential as an efficient and low-cost diagnostic reagent. In this study, the DNA of the PRV SC strain was used as the template, and the recombinant fragment of gI (633 bp) was amplified via PCR using synthetic primers, and was then ligated into the pET-30a expression vector. The constructs were transferred into () for prokaryotic expression, and the antigenicity of the expression products was identified by Western blot analysis with pig positive serum against PRV. The recombinant protein was purified by a Ni column, and BALB/c mice were immunized with purified gI protein to obtain anti-gI-positive serum. After PK-15 cells had been infected by PRV for 48 h, the immunogenicity of purified gI protein was identified with a fluorescence immunoassay using anti-gI mouse serum. The recombinant plasmid (pET-30a-gI) was expressed, and the native gI protein was obtained after denaturation by urea and renaturation by dialysis. A small-scale ELISA test containing 1.0 µg/mL of purified gI protein was designed to evaluate pig serum (80 samples), and the results of the ELISA test were compared to those of competitive ELISA (cELISA) tests using IDEXX Kits, which resulted in 97.5% consistency. The results suggested that the truncated gI protein may be a potential diagnostic reagent.
血清学检测是诊断伪狂犬病病毒（PRV）感染的重要方法。我们旨在研究PRV的包膜糖蛋白I（gI），一种强免疫原，及其作为高效低成本诊断试剂的潜力。在本研究中，以PRV SC株的DNA为模板，使用合成引物通过PCR扩增gI的重组片段（633 bp），然后将其连接到pET-30a表达载体中。构建体被转入（）进行原核表达，并用猪抗PRV阳性血清通过蛋白质免疫印迹分析鉴定表达产物的抗原性。重组蛋白通过镍柱纯化，并用纯化的gI蛋白免疫BALB/c小鼠以获得抗gI阳性血清。PRV感染PK-15细胞48小时后，使用抗gI小鼠血清通过荧光免疫测定法鉴定纯化的gI蛋白的免疫原性。表达重组质粒（pET-30a-gI），经尿素变性和透析复性后获得天然gI蛋白。设计了一个含有1.0 µg/mL纯化gI蛋白的小规模ELISA试验来评估猪血清（80份样品），并将ELISA试验结果与使用IDEXX试剂盒的竞争ELISA（cELISA）试验结果进行比较，一致性为97.5%。结果表明，截短的gI蛋白可能是一种潜在的诊断试剂。
