TLR4^-/- DF1细胞系感染NDV后免疫机制的初步研究  

Construction of TLR4^-/- DF1 cell line by CRISPR/Cas9 and preliminary mechanistic study on the host immune responses to Newcastle disease virus infection

作　　者：王辉 鲁国涛 许曼 曾为俊 邵玉乐 赵丽丽[2] 陈洪岩[2] 刘建华[1] 孟庆文[2] WANG Hui;LU Guo-tao;XU Man;ZENG Wei-jun;SHAO Yu-le;ZHAO Li-li;CHEN Hong-yan;LIU Jian-hua;MENG Qing-wen(College of Animal Medicine,Xinjiang Agricultural University,Urumqi 830000,China;State Key Laboratory of Veterinary Biotechnology,Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830000 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/黑龙江省实验动物与比较医学重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2019年第12期1244-1250,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室自主研究课题(SKLVBP201801);转基因生物新品种培育重大专项(2009ZX08006-001B)。

摘　　要：为初步探究鸡TLR4在抗病毒感染天然免疫中发挥的作用,本研究利用CRISPR/Cas9基因编辑技术针对鸡TLR4基因的功能域,设计、构建了CRISPR/Cas9双质粒表达系统,转染鸡DF1细胞;经流式细胞仪分选、测序及western blot鉴定筛选得到TLR4^-/- DF1细胞系;利用新城疫病毒(NDV)感染TLR4^-/- DF1细胞系,采用绝对荧光定量PCR检测NDV拷贝数,相对荧光定量PCR检测TLR4下游接头蛋白、细胞因子及干扰素基因的转录水平。结果显示:与野生型DF1细胞相比,NDV感染6 h、16 h和36 h的TLR4^-/- DF1细胞中病毒基因拷贝数显著升高(p<0.05),16 h升高了2.95倍,感染6 h、16 h和24 h下游接头蛋白MyD88基因转录水平下降,感染16 h促炎因子IL-1β下降了48.5%(p<0.01),IL-8下降了62.5%(p<0.01),I型干扰素转录水平下降。以上结果表明:NDV感染诱导的天然免疫应答可能是通过MyD88途径诱导高水平促炎性因子和I型干扰素(IFN-α、IFN-β)的表达,从而抑制NDV的增殖,TLR4在NDV感染引起的天然免疫应答和疾病防控研究中具有重要意义。同时,本研究TLR4^-/- DF1细胞系的建立可用于病毒引起的炎性因子风暴及天然免疫抗病毒作用的探究。In order to explore the role of chicken TLR4 in the antiviral innate immunity,present study constructed TLR4^-/- DF1 cell line by transferring a CRISPR/Cas9 double plasmid expression system targeting the functional domain of chicken TLR4 gene into avian DF1 cells.Cells were isolated by flow cytometry sorting and the knockout of TLR4 gene was validated by sequencing and western blot.Using TLR4^-/- DF1 cells infected with Newcastle disease virus(NDV)as model,the replication of NDV and the transcription of TLR4 downstream adaptor proteins,cytokines and interferons were assayed by absolute fluorescent quantitative PCR and real-time PCR,respectively.Comparing with those of wide type DF1 cells,the viral copy number was increased significantly upon knockout of TLR4 gene at 6,16,and 36 hours post infection(hpi),which was increased 2.95 times at 16 hpi(p<0.05).The transcription of TLR4 downstream protein MyD88 was decreased significantly upon knockout of TLR4 gene at 6,16,and 24 hpi(p<0.05).The transcription of pro-inflammatory factors IL-1β was decreased 48.5%(p<0.01)and IL-8 was decreased 62.5%(p<0.01)at 16 hpi.The expression of type I interferons was also decreased significantly upon knockout of TLR4 gene(p<0.05).Overall,present study suggests that host cells may combat NDV by triggering high expression of pro-inflammatory factors and type I interferons(IFN-α and IFN-β)via MyD88-mediated pathways,and TLR4 may play an important role in this process.Besides,the TLR4^-/- DF1 cell line constructed in present study can be widely used for the studies of viral-induced inflammatory storms and antiviral innate immune responses.

关 键 词：TLR4 CRISPR/Cas9系统 DF1 基因敲除 NDV 

分 类 号：S858.31[农业科学—临床兽医学]