Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University , Tai'an, China .
Viral Immunol. 2018 Jul/Aug;31(6):407-416. doi: 10.1089/vim.2017.0170. Epub 2018 Apr 26.
This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens against ALV-J infection. This study first reported the methods on preparing the secretory recombinant ALV-J gp85 protein using P. pastoris and evaluated its immunoprotection.
本研究旨在利用毕赤酵母（Pichia pastoris）制备分泌型重组禽白血病病毒（ALV-J）J 亚群 gp85 蛋白，并结合 CpG-ODN 佐剂评估其作为疫苗抗原的免疫保护作用。设计并构建了含有 ALV-J gp85 基因的分泌型重组质粒 pPIC9-gp85，转染毕赤酵母（GS115）细胞基因组。甲醇诱导表达重组质粒，细胞培养上清中的表达产物经内切糖苷酶消化鉴定和单克隆抗体（MAb）JE9 介导的 Western blot 鉴定。纯化后的产物与 CpG-ODN 佐剂结合，7 日龄雏鸡肌肉注射，首免后 21 天（dpi）、42dpi 和 56dpi 进行 3 次加强免疫。攻毒后检测抗体和细胞免疫应答，并分析保护效果。结果表明，成功构建了分泌型 pPIC9-gp85 质粒，可在 GS115 细胞中稳定表达。表达产物为 N-乙酰葡萄糖基化，可特异性结合 MAb（JE9）。与 CpG-ODN 佐剂结合的分泌型 gp85 蛋白可诱导更高的抗体应答和脾淋巴细胞增殖应答及 IFN-γ诱导应答，并可保护所有接种鸡免受 ALV-J 攻毒引起的病毒血症和免疫抑制损伤。体外中和试验结果表明，具有一定 ALV-J 抗体滴度的抗血清可中和 ALV-J 株，抑制病毒在体外生长。IFA 结果表明，抗血清中的 IgG 抗体可特异性结合细胞中的 ALV-J 株。综上所述，成功制备了分泌型重组 gp85 蛋白，作为一种新型乙酰葡萄糖基化 gp85 蛋白，与 CpG-ODN 佐剂结合可保护接种鸡免受 ALV-J 感染。本研究首次报道了利用毕赤酵母制备分泌型重组 ALV-J gp85 蛋白的方法，并对其免疫保护作用进行了评价。
