稳定表达3种猪腹泻病毒嵌合抗原的CHO细胞系构建与鉴定  

Construction and identification of CHO cell line stably expressing chimeric antigens of three porcine diarrhea viruses

作　　者：宋旭 赵永祥 范宝超[1] 郭容利[1] 李基棕[1] 刘静[2] 李彬[1,2] SONG Xu;ZHAO Yongxiang;FAN Baochao;GUO Rongli;LI Jizhong;LIU Jing;LI Bin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]河北农业大学动物医学院,河北保定071001

出　　处：《畜牧与兽医》2023年第3期104-109,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2022YFD1800801);国家自然科学基金(32002283);江苏省农业自主创新基金(CX[21]3139)。

摘　　要：为构建稳定表达猪流行性腹泻病毒(PEDV)、猪胃肠炎病毒(TGEV)和猪Delta冠状病毒(PDCoV)主要抗原的CHO细胞系,为猪病毒性腹泻三联疫苗的研发提供基础材料,本研究将PEDV的S基因的COE区域、TGEV和PDCoV的S基因的受体结合区域(RBD),通过同源重组的方式进行融合,克隆到慢病毒表达质粒pLV-EF1a-IRES-Puro中,包装得到重组慢病毒。使用重组慢病毒感染CHO细胞,经过嘌呤霉素和单克隆细胞筛选,基因水平和蛋白水平表达验证,成功构建了稳定表达3种猪腹泻病毒抗原融合蛋白的CHO细胞系,为进一步研制PEDV-TGEV-PDCoV的亚单位疫苗奠定了基础。The aim of this study was to construct a CHO cell line that stably expresses the main antigens of porcine epidemic diarrhea virus(PEDV), porcine gastroenteritis virus(TGEV) and porcine Delta coronavirus(PDCoV), which would provide basic materials for the development of a triple vaccine for porcine viral diarrhea. The COE region of the S gene of PEDV, the receptor-binding region(RBD) of the S gene of TGEV and PDCoV were fused by homologous recombination, and they cloned into the lentiviral expression plasmid pLV-EF1a-IRES-Puro, and were then packaged to obtain recombinant lentivirus. CHO cells were infected with the recombinant lentivirus, and were screened with puromycin. Monoclonal CHO cells were obtained after several screening cycles. The expression of the chimeric gene in the screened CHO cells was certificated at the gene and protein levels. The results demonstrated that we successfully constructed a CHO cell line that stably expressed three-viral antigen fusion protein, laying a foundation for the further development of PEDV-TGEV-PDCoV subunit vaccine.

关 键 词：CHO细胞系 稳定表达 仔猪腹泻病毒 慢病毒 

分 类 号：S855.3[农业科学—临床兽医学]