牛冠状病毒N蛋白的原核表达及对小鼠免疫原性的初步评价  

Prokaryotic expression of bovine coronavirus N protein and preliminary evaluation of its immunological effect on mice

作　　者：蒋倩 闵芳 马志刚 梁宇萌 陶芯宇 丁晓军 李添庆 钟旗[2] 姚刚[1] 马雪连 JIANG Qian;MIN Fang;MA Zhigang;LIANG Yumeng;TAO Xinyu;DING Xiaojun;LI Tianqing;ZHONG Qi;YAO Gang;MA Xuelian(Xinjiang Key Laboratory of New Drug Study and Creation for Herbivorous Animals,College of Veterinary Medicine,Xinjiang AgriculturalUniversity,Urumqi 830052,China;Institute of Animal Science,Xinjiang Academy of AnimalSciences,Urumqi 830011,China)

机构地区：[1]新疆农业大学动物医学学院新疆草食动物新药研究与创制重点实验室,新疆乌鲁木齐830052 [2]新疆畜牧科学院动物科学研究所,新疆乌鲁木齐830011

出　　处：《中国兽医学报》2024年第12期2540-2548,共9页Chinese Journal of Veterinary Science

基　　金：新疆维吾尔自治区创新团队基金资助项目(2023D14018);新疆维吾尔自治区重点学科和重点实验室开放课题资助项目(XJCDVMM-HDRC-S202301);新疆维吾尔自治区重大专项资助项目(2021A02003-1)。

摘　　要：牛冠状病毒(bovine coronavirus,BCoV)可引起新生犊牛腹泻和呼吸道疾病,严重时可导致犊牛死亡,给养牛业造成巨大的经济损失。目前我国尚未有自主研发的针对BCoV的疫苗,导致BCoV流行性高、传播范围广,因此开发具有保护性BCoV的疫苗是当务之急。本研究旨在表达BCoV N蛋白并分析其免疫原性,首先用PCR方法扩增N蛋白基因,构建pET-30a-N重组质粒,表达并纯化N蛋白,再将N蛋白与佐剂配伍免疫小鼠;采用间接ELISA检测小鼠IgG和IgA抗体水平及滴度,利用流式细胞术检测小鼠脾脏T淋巴细胞分群比例以及相关免疫细胞因子的释放情况。结果显示,成功获得BCoV可溶性N蛋白,大小约为55kDa;ELISA检测结果显示,免疫组小鼠血清IgG抗体效价为1∶51200,免疫组小鼠血清IgA抗体效价为1∶3200;流式细胞术检测结果显示,与对照组相比,免疫组CD4+/CD8+T淋巴细胞亚群比例极显著升高(P<0.01),TNF-α释放显著增多(P<0.05),产生偏向辅助性T细胞增加和TNF-α分泌增多的细胞免疫应答。研究表明,通过原核表达系统能成功实现BCoV N蛋白的可溶性表达,且获得的BCoV N蛋白免疫原性良好,可诱导免疫小鼠产生强烈的体液免疫和细胞免疫反应。为开发安全有效的BCoV亚单位疫苗提供了重要的技术支持。Bovine coronavirus(BCoV)can cause diarrhea in calves,as well as respiratory diseases,and lead to the death of calves in severe cases,resulting in great economic losses to the cattle production.BCoV is highly prevalent and widely spread.So far in China there is no independently developed vaccine against BCoV on the market.In this study,the most conserved N gene of BCoV was first selected to construct a prokaryotic expression vector,and the N protein was expressed and purified;subsequently,BALB/c mice were immunized with BCoV N protein combined with Freund's adjuvant,and the antibody potency,the proportion of splenic T-lymphocyte subpopulation and the cytokine release of the immunized mice were detected by ELISA and flow cytometry.The results showed that a soluble BCoV N protein about 55kDa in size was successfully obtained.The ELISA test showed that the antibody potency of serum IgG and IgA was 1∶51200and 1∶3200,respectively,in mice immunized with BCoV N protein combined with Freund's adjuvant.The flow cytometry test showed that the percentage of CD4+/CD8+T-lymphocyte subsets was extremely higher in the immunized mice(P<0.01),and the release of TNF-αwas significantly higher(P<0.05),producing a cellular immune response biased towards the Th1type in comparison with that in control mice.It is indicated that the soluble expression of BCoV N protein can be successfully achieved by the prokaryotic expression system,and the obtained BCoV N protein presents good immunogenicity which induces strong humoral,mucosal and cellular immune responses in the immunized mice.Our study provides an important technical support for the development of a safe and effective BCoV subunit vaccine.

关 键 词：牛冠状病毒 N蛋白 亚单位疫苗 小鼠 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]