猪流行性腹泻病毒细胞适应毒LJB-03株在三种Vero细胞上增殖效果的比较  

Comparison of effects of cell-adapted porcine epidemic diarrhea virus strain LJB-03 proliferated on three kinds of Vero cells

作　　者：郭卫龙 解伟纯 侯星宇 李智力 姜艳平[1] 崔文[1] 王丽[1] 乔薪瑗[1] 周晗[1] 徐义刚[1] 李一经[1] 唐丽杰[1] GUO Wei-long;XIE Wei-chun;HOU Xing-yu;LI Zhi-li;JIANG Yan-ping;CUI Wen;WANG Li;QIAO Xin-yuan;ZHOU Han;XU Yi-gang;LI Yi-jing;JANG Li-jie(College of Veterinary Medicine,Northeast Agriculturcd University,Harbin 150030,China;Shanghai Basal Media Technologies Co.,LTD.,Shanghai 201499,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]上海源培生物科技股份有限公司,上海201499

出　　处：《中国兽医科学》2019年第3期301-308,共8页Chinese Veterinary Science

基　　金："十三五"国家重点研发计划项目(2017YFD0501105);国家自然科学基金资助项目(31472226)

摘　　要：为探究猪流行性腹泻病毒(PEDV)细胞适应毒LJB-03株在3种Vero细胞上的增殖特性,分别以正常Vero细胞、适应无血清培养基的Vero细胞和稳定表达跨膜丝氨酸蛋白酶2 (TMPRSS2)的Vero/TMPRSS2细胞(繁殖PEDV时无须添加胰酶)为宿主细胞,繁殖PEDV LJB-03株,观察这3种细胞接毒后的病变特点和病变时间,利用空斑法比较其增殖特性,并以间接免疫荧光和电镜技术鉴定病毒的增殖。结果表明,PEDV LJB-03株在正常Vero细胞和适应无血清培养基的Vero细胞上增殖时需添加的胰酶最佳质量浓度为7ug/m L;在这3种细胞上增殖时的最佳感染复数均为0.01;PEDV LJB-03株在3种细胞上效价达到峰值的时间均为36 h,其中在正常Vero细胞上病毒效价最高为7.17×10~7PFU/m L,在Vero/TMPRSS2细胞上病毒效价为3.33×10~7PFU/m L,在无血清Vero细胞上的病毒效价为2.85×10~7PFU/m L。综上所述,正常Vero细胞上PEDV LJB-03株的病毒效价可达其他2种细胞的2倍以上,但在培养病毒时正常Vero细胞需添加胰酶和血清,因此,适应无血清培养基的Vero细胞与Vero/TMPRSS2细胞在培养PEDV LJB-03株时更具有潜力。In order to compare the proliferation characteristics of porcine epidemic diarrhea virus(PEDV)LJB-03 strain on 3 kinds of Vero cells,PEDV LJB-03 strain was propagated on the normal Vero cells,the Vero cells adapted to serum-free medium,and the Vero/TMPRSS2 cells stably expressing transmembrane serine protease 2(TMPRSS2)without the addition of trypsin.The characteristics and time of cytopathic effect of these three kinds of cells were observed,then the proliferation characteristics of the virus were analyzed by plaque assay.The virus was identified by indirect immunofluorescence assay and electron microscopy.The results showed that the optimal concentration of trypsin was 7ug/m L when LJB-03 strain was proliferated in the normal Vero and the Vero cells adapted to serum-free medium;the best multiplicity of infection(MOI)were both 0.01 when proliferated on the three kinds of cells.The virus titer reached peak at 36 hours post infection.Among these peaks,the virus titer on the normal Vero cells was the highest,which was 7.17×10^7 PFU/m L;followed by Vero/TMPRSS2 cells,the virus titer reached 3.33×10^7 PFU/m L;virus titer in the Vero cells adapted to serum-free medium was 2.85×10^7 PFU/m L.In summary,the virus titer of PEDV LJB-03 strain on the normal Vero cells was 2 times higher than the other two cells,but serum and trypsin were needed when proliferating this virus on the normal Vero cells.Therefore,the Vero cells adapted to serum-free medium and the Vero/TMPRSS2 cells have more potential in the propagation of PEDV LJB-03 strain.

关 键 词：猪流行性腹泻病毒 细胞适应毒 VERO细胞 增殖 

分 类 号：S852.659.6[农业科学—基础兽医学]