口蹄疫病毒样颗粒诱导表达型载体的构建及其BHK-21细胞池的筛获  

Construction of foot-and-mouth disease virus like particlesinduced expression vectors and screening of BHK-21 cell pools

作　　者：谭书桢 董虎[1] 孙世琪[1] 郭慧琛[1,2,3] TAN Shuzhen;DONG Hu;SUN Shiqi;GUO Huichen(State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,Gansu,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Yunnan Provincial Key Laboratory of Tropical and Subtropical Animal Viral Diseases,Yunnan Academy of Animal Husbandry and Veterinary Sciences,Kunming 650224,Yunnan,China)

机构地区：[1]中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州730000 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]云南省畜牧兽医科学院云南热带亚热带动物病毒重点病实验室,云南昆明650224

出　　处：《生物工程学报》2023年第12期4849-4860,共12页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2021YFD1800300,2022YFD1800603);国家自然科学基金(32072847,32072859,32002272);科技人才与平台计划(202205AF150007)。

摘　　要：瞬时表达是目前利用哺乳动物细胞表达口蹄疫病毒(foot-and-mouth disease virus,FMDV)衣壳蛋白的主流方法。为实现染色体稳定表达FMDV衣壳蛋白并高效组装出病毒样颗粒(virus like particles,VLPs),本研究构建了piggy Bac(PB)转座-组成型表达、PB转座-四环素(tetracycline,Tet)诱导型表达两套质粒。利用荧光蛋白标记技术,验证了质粒的功能。通过抗生素筛选得到了组成型表达P12A3C(WT/L127P)基因的BHK-21细胞池(C-WT、C-L127P)和诱导型表达P12A3C(WT/L127P)基因的BHK-21细胞池(I-WT、I-L127P)。荧光观察和PCR检测证明了绿色荧光蛋白、3C蛋白酶、反向四环素转录激活因子等基因的稳定整合。Western blotting、酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)实验证明了细胞池I-L127P具有更强的衣壳蛋白和VLPs生产能力。本研究首次实现了哺乳动物细胞染色体诱导表达FMDV衣壳蛋白,有助于推动哺乳动物生产FMDV VLPs疫苗的技术工艺,也为构建其他蛋白的哺乳动物细胞诱导型表达系统提供了参考。Transient expression is the major method to express foot-and-mouth disease virus(FMDV)capsid proteins in mammalian cells.To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles(VLPs)in cells,the plasmids of piggyBac(PB)transposon-constitutive expression and PB transposon-tetracycline(Tet)inducible expression vectors were constructed.The function of the plasmids was tested by fluorescent proteins.By adding antibiotics,the constitutive cell pools(C-WT,C-L127P)expressing P12A3C(WT/L127P)genes and the inducible cell pools(I-WT,I-L127P)expressing P12A3C(WT/L127P)genes were generated.The genes of green fluorescent protein,3C protease and reverse tetracycline transactivator(rtTA)were integrated into chromosome,which was confirmed by fluorescence observation and PCR testing.The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs,which was confirmed by Western blotting and enzyme linked immunosorbent assay(ELISA),respectively.In conclusion,inducing the chromosomal expression of FMDV capsid proteins was firstly reported,which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.

关 键 词：口蹄疫病毒 病毒样颗粒 质粒构建 细胞池 诱导表达 

分 类 号：S852.65[农业科学—基础兽医学]