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
肖丽 1赵淑庆 2袁雪松 3范丽原 3易鑫 4陈琢琦 4李彬 5李基棕 5主性6
作者信息
- 1. 贵州大学动物科学学院,贵州 贵阳 550025;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
- 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;南京工业大学 药学院,江苏南京 210000
- 3. 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;南京农业大学动物医学院,江苏南京 210095
- 4. 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
- 5. 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;南京农业大学动物医学院,江苏南京 210095;江苏大学生命科学学院/食品与生物工程学院,江苏 镇江 212013
- 6. 贵州大学动物科学学院,贵州 贵阳 550025
- 折叠
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.
Abstract
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)express-ing 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 lenti-virus 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 de-tected 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 293T cell line stably expressing PDCoV-S and RBD proteins was successfully constructed,and the recombi-nant 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∶128 and 1∶64,respectively.In summary,the 293T cell line constructed in this study can stably express PD-CoV-S and RBD proteins,which lays a foundation for further development of PDCoV subunit vac-cine.
关键词
293T细胞系/PDCoV/慢病毒/稳定表达Key words
293T cell line/PDCoV/lentivirus/stable expression引用本文复制引用
基金项目
国家重点研发计划(十四五)(2022YFD1800803)
江苏省自然科学基金(BK20221432)
出版年
2024
NETL
NSTL
万方数据