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

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.