稳定表达PDCoV-S和RBD蛋白的293T细胞系构建与免疫原性评价  

Construction and immunogenicity evaluation of 293T cell linesexpressing PDCoV-S and RBD proteins

作　　者：肖丽 赵淑庆 袁雪松 范丽原 易鑫 陈琢琦 李彬[2,3,4] 李基棕 主性[1] XIAO Li;ZHAO Shuqing;YUAN Xuesong;FAN Liyuan;YI Xin;CHEN Zhuoqi;LI Bin;LI Jizong;ZHU Xing(College of Animal Science,Guizhou University,Guiyang 550025,China;Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;School of Food and Biological Engineering/School of Life Sciences,Jiangsu University,Zhenjiang,Jiangsu 210013,China;School of Pharmacy,Nanjing Tech University,Nanjing 210000,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室,江苏南京210014 [3]南京农业大学动物医学院,江苏南京210095 [4]江苏大学生命科学学院/食品与生物工程学院,江苏镇江212013 [5]南京工业大学药学院,江苏南京210000

出　　处：《中国兽医学报》2024年第5期959-965,共7页Chinese Journal of Veterinary Science

基　　金：“十四五”国家重点研发计划资助项目(2022YFD1800803);江苏省自然科学基金资助项目(BK20221432)。

摘　　要：为构建稳定表达猪δ冠状病毒(PDCoV)S和RBD蛋白的293T细胞系,获得PDCoV S和RBD蛋白。本研究将优化合成的PDCoV S蛋白全长基因及其RBD的表达质粒进行双酶切鉴定,将重组质粒pLV-S-Puro、pLV-RBD-Puro、psPRX2、pMD2.G同时转染293T细胞中进行慢病毒包装,收集上清感染293T细胞,经嘌呤霉素初筛得到的多细胞克隆,进一步通过终点稀释法筛选获得稳定表达PDCoV S和RBD蛋白的单克隆293T细胞系,通过Western blot检测蛋白表达,用纯化的PDCoV S和RBD蛋白分别免疫BALB/c小鼠,对重组蛋白进行免疫原性检测。结果表明,稳定表达PDCoV S和RBD蛋白的293T细胞系构建成功,获得了重组慢病毒,纯化PDCoV S和RBD重组蛋白均可以诱导小鼠产生较高的IgG抗体(S:1.2;RBD:0.9),中和抗体水平分别达1∶128和1∶64。本研究构建的293T细胞系能稳定表达PDCoV S和RBD蛋白,为进一步研制PDCoV亚单位疫苗奠定了基础。This study aims to construct a 293T cell line stably expressing porcine deltacoronavirus(PDCoV)S and RBD proteins and provide basic materials for the development of PDCoV subunit vaccine.The full-length gene of PDCoV-S protein and its receptor binding domain(RBD)expressing plasmid were identified by double enzyme digestion.The recombinant plasmids pLV-S-Puro,pLV-RBD-Puro,psPRX2,and pMD2.G were simultaneously transfected into 293T cells for lentivirus packaging.The supernatant was collected to infect 293T cells,and the polyclonal cells were obtained by puromycin screening.Then,the monoclonal 293T cell line stably expressing PDCoV S and RBD proteins was screened by end-point dilution method,and the protein expression was detected by Western blot.BALB/c mice were immunized with purified S and RBD proteins,and the immunogenicity of the recombinant proteins was detected.The results showed that the 293Tcell line stably expressing PDCoV-S and RBD proteins was successfully constructed,and the recombinant lentivirus was obtained.The purified S and RBD recombinant proteins could induce mice to produce higher IgG antibodies(S:1.2;rBD:0.9),and neutralizing antibodies were 1∶128and 1∶64,respectively.In summary,the 293Tcell line constructed in this study can stably express PDCoV-S and RBD proteins,which lays a foundation for further development of PDCoV subunit vaccine.

关 键 词：293T细胞系 PDCoV 慢病毒 稳定表达 

分 类 号：S852.65[农业科学—基础兽医学]