南非2型口蹄疫病毒VP1基因的原核可溶性表达及免疫原性分析  

Prokaryotic Soluble Expression and Immunogenicity Analysis of VP1 Gene of Foot-and-mouth disease virus serotypes SAT2

作　　者：李国秀 欧云文 丁耀忠[1] 李茜 代军飞 刘磊 张杰[1] LI Guo-Xiu;OU Yun-Wen;DING Yao-Zhong;LI Qian;DAI Jun-Fei;LIU Lei;ZHANG Jie(Office International Des Epizootics(OIE)/National Foot-and-Mouth Disease(FMD)Reference Laboratory/State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Animal Disease Prevention and Control Center of Kaijiang County,Dazhou 636250.China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou.730070,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室/世界动物卫生组织(OIE)/国家口蹄疫参考实验室,兰州730046 [2]甘肃农业大学动物医学院,兰州730070 [3]开江县动物疫病预防控制中心,达州636250

出　　处：《农业生物技术学报》2020年第10期1862-1869,共8页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2017YFD0501800;2016YFD0501500);甘肃省国际科技特派员项目(17JR7WA030);中国农业科学院基本科研业务费;四川省科技计划资助项目(2019JDRC0110;2020JDRC0146)。

摘　　要：南非2型口蹄疫(Foot-and-mouth disease serotypes Southern African territories 2, SAT2-FMD)是由南非2型口蹄疫病毒(Foot-and-mouth disease virus serotypes Southern African territories 2, SAT2-FMDV)引起偶蹄动物的一种急性、热性、高度接触性传染病。当前全球贸易和人员往来频繁,SAT2-FMD传入中国的风险极高,这将严重威胁我国养殖业持续健康发展。为原核可溶性表达SAT2-FMDV (GenBank No.JX014256) VP1基因,探索VP1融合蛋白的免疫原性,本研究以序列优化的pUC57-His-SUMO-SAT2-VP1重组质粒为模板,设计特异性引物,PCR扩增获得993 bp目的序列,扩增产物克隆入pET32a (+)原核表达载体,构建pET32a-HisSUMO-VP1重组质粒,转入大肠杆菌(Escherichia coli) BL21(DE3),诱导表达,亲和层析纯化。将纯化后HisSUMO-VP1融合蛋白与弗氏佐剂混匀乳化,免疫Balb/C小鼠(Mus musculus),并对免疫小鼠血清抗体、脾淋巴细胞增殖和细胞因子水平进行测定。研究结果表明,HisSUMO-VP1融合蛋白实现原核可溶性表达,大小约为63 kD,且具有较好的反应原性;免疫小鼠实验结果发现,HisSUMOVP1融合蛋白组的小鼠免疫血清抗体水平极显著高于HisSUMO蛋白组和PBS组(P<0.001),且抗体水平呈上升趋势;HisSUMO-VP1融合蛋白组小鼠脾脏淋巴细胞发生显著地增殖反应(P<0.001),细胞因子IFN-γ和IL-2水平显著高于PBS组(P<0.05)。本研究成功实现SAT2-FMDV-VP1基因的原核可溶性表达,表达的HisSUMO-VP1融合蛋白具有良好的免疫原性,为SAT2-FMDV疫苗及检测方法的研发提供数据支持。Foot-and-mouth disease serotypes Southern African Territories 2(SAT2-FMD) is an acute, heat,and highly contactive infectious disease caused by Foot-and-mouth disease virus serotypes Southern African Territories 2(SAT2-FMDV). At present, global trade and mutual visits are frequent, and the risk of SAT2-FMD entering China is extremely high, which will seriously threaten the sustainable and healthy development of Chinese breading industry. This experiment was aimed to prokaryotic soluble expression and analysis the immunogenicity of VP1 gene of SAT2-FMDV(GenBank No. JX014256). The gene was amplified from the recombined plasmid pUC57-His-SUMO-SAT2-VP1 by PCR, with a product of approximately 993 bp. The identified recombined plasmid pET32 a-HisSUMO-VP1 was expressed by Escherichia coli BL21(DE3) and was induced by 1 mmol/L isopropy-β-D-thiogalactoside(IPTG). The HisSUMO-VP1 protein was purified by Ni-NTA, and the purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDA-PAGE) and Western blot. The HisSUMO-VP1 protein was used as an immunogen for immunization of Balb/C mice(Mus musculus), and the serum antibody, spleen lymphocyte proliferation and cytokine levels were detected. The results of SDA-PAGE and Western blot showed that the HisSUMO-VP1 protein with a relative molecular weight of 63 k D was highly soluble and had good reactivity. The immune experiment found that the serum antibody levels in the immunized mice vaccinated with the HisSUMO-VP1 fusion protein group was significantly higher than those in the HisSUMO protein vaccinated group and the mock group(P<0.001), and the mice spleen lymphocytes had a significant lymphocyte proliferation response(P<0.001). The levels of IFN-γ and IL-2 were significantly higher than those in the PBS group(P<0.05). The HisSUMO-VP1 protein with excellent immunogenicity is solublely expressed in E. coli, providing data support for the diagnosis and vaccine development of SAT2-FMDV.

关 键 词：口蹄疫病毒(FMDV) 南非2型(SAT2) VP1基因 可溶性表达 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]