敲除RIG-I基因的PK-15细胞系的建立及RIG-I对猪圆环病毒2型感染的作用  

ESTABLISHMENT OF PK-15 CELL LINE VIA KNOCKOUT OF RIG-Ⅰ GENE AND EVALUATION OF PORCINE CIRCOVIRUS TYPE 2 INFECTION

作　　者：许曼 黄立平[2] 邵玉乐 夏德利[2] 曾为俊 王辉 鲁国涛 刘长明[2] 陈洪岩[2] 王金泉[1] 孟庆文[2] XU Man;HUANG Li-ping;SHAO Yu-le;XIA De-li;ZENG Wei-jun;WANG Hui;LU Guo-tao;LIU Chang-ming;CHEN Hong-yan;WANG Jin-quan;MENG Qing-wen(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Heilongjiang Provincial Key Laboratory of Laboratory Animals and Comparative Medicine,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,CAAS,Harbin 150069,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [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,哈尔滨150069

出　　处：《中国动物传染病学报》2019年第5期14-21,共8页Chinese Journal of Animal Infectious Diseases

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：利用CRISPR/Cas9基因编辑技术构建敲除RIG-I基因的PK-15细胞,初步探究视黄酸诱导基因I(retinoic acid-inducible gene-I,RIG-I)对猪圆环病毒2型(Porcine circovirus type 2,PCV2)感染的作用。本研究根据CRISPR/Cas9靶点设计原则,设计了针对猪RIG-I基因的2条sgRNA,构建重组载体pUC19-RIG-I-sgRNA1和pUC19-RIG-I-sgRNA2,转染PK-15细胞,通过流式细胞仪分选单细胞、测序、Western blot检测RIG-I敲除情况。PCV2感染细胞后,通过荧光定量PCR分析RIG-I及其下游分子mRNA水平,IPMA方法及TaqMan定量PCR检测病毒增殖水平。结果显示,筛选出的1株RIG-I基因缺失37 bp的PK-15细胞,Western blot检测RIG-I蛋白不表达。PCV2感染正常PK-15细胞48 h和72 h后,RIG-I mRNA水平上调,表明PCV2可激活RIG-I。敲除RIG-I基因,可下调MAVS、STING、IRF3、IFN-β的mRNA水平,抑制PCV2在PK-15细胞中复制,初步表明RIG-I信号通路促进PCV2在PK-15细胞中复制。本研究为PCV2感染的天然免疫机制研究提供了良好的细胞模型。The RIG-I gene of PK-15 cell line was knocked out by CRISPR/Cas9 editing technology to investigate its effect on porcine circovirus type 2(PCV2)infection.Based on the principle of CRISPR/Cas9 target,two sgRNAs targeting to delete pig RIG-I gene were designed and recombinant plasmid was constructed for transfection of PK-15 cells.Transfected PK-15 cells were sorted out by flow cytometry and knocked out was examined by sequencing and Western blotting.The knockout PK-15 cells were infected with PCV2 and analyzed by real-time PCR for expression of mRNA levels of RIG-I and its downstream molecules.The virus proliferation level was detected by IPMA method and TaqMan quantitative PCR.The results showed that one strain of RIG-I gene was deficient in 37 bp PK-15 cells,and the expression of RIG-I protein was not detected in Western blotting.At 48 h and 72 h post PCV2 infection,RIG-I mRNA level was up-regulated in PK-15 cells,indicating that PCV2 activated RIG-I and knockdown of RIG-I gene might down-regulate mRNA levels of MAVS,STING,IRF3 and IFN-β.Inhibition of PCV2 replication in knockout PK-15 cells indicated a promotion effect of RIG-I signaling pathway.This study provided a valuable cell model for the study of the mechanism of natural immune regulation triggered by PCV2 infection.

关 键 词：CRISPR/Cas9基因编辑 视黄酸诱导基因Ⅰ信号通路 PK-15 猪圆环病毒2型 

分 类 号：S852.659.2[农业科学—基础兽医学]