摘要
禽流感病毒(AIV)能劫持宿主ANP32A蛋白与流感病毒聚合酶结合,促进AIV复制,但chANP32A调节流感病毒复制的分子机制尚存在诸多未知。本研究利用腺病毒介导的CRISPR/Cas9基因编辑技术,靶向chANP32A 129位氨基酸(AA)设计、合成了sgRNA序列,构建Ad-SpCas9腺病毒载体、包装腺病毒。以重组腺病毒感染的DF-1细胞经亚克隆培养、PCR扩增和基因组测序,鉴定到3株缺失6、21、24个碱基的细胞株。以H9N2禽流感病毒感染细胞,RT-qPCR结果显示,KO-6、KO-21、KO-24细胞株的病毒基因组RNA拷贝数显著降低;TCID50测定病毒生长曲线,病毒滴度显著下降;流感病毒双荧光素酶报告系统测定聚合酶活性,结果显示缺失细胞株流感病毒聚合酶活性降低30~3000倍。研究发现,KO-6、KO-21细胞株抑制AIV复制,KO-24细胞株显著抑制AIV复制,缺失chANP32A 125~132 AA更显著抑制AIV复制。本研究为chANP32A基因功能研究以及研发具有抗禽流感病毒且chANP32A生物学功能不改变的基因编辑鸡奠定了基础。
Avian influenza virus(AIV)can hijack the host ANP32A protein to bind to influenza virus polymerase and promote AIV replication.However,the molecular mechanism by which ch ANP32A regulates influenza virus replication is still unknown.In this study,we designed and synthesised sg RNA sequences targeting amino acid(AA)129 of ch ANP32A using adenovirus-mediated CRISPR/Cas9 gene editing,constructed Ad-Sp Cas9 adenoviral vector,and packaged adenovirus.The recombinant adenovirus was used to infect DF-1 cells through subclonal culture,PCR amplification and genome sequencing,and three cell lines with deletion of 6,21 and 24 bases were identified.Cells were infected with H9N2 avian influenza virus and measured by RT-q PCR,TCID_(50),and influenza virus polymerase activity methods,RT-q PCR results showed that the viral genome RNA copy number of the KO-6,KO-21,and KO-24 cell lines was significantly reduced,the viral growth curves were measured by TCID50,and the viral titres were significantly reduced,the influenza virus dual luciferase reporter.The results showed that the viral polymerase activity of influenza virus in the deletion cell lines was reduced by 30—3000 times.It was found that KO-6 and KO-21 cell lines inhibited AIV replication,KO-24 cell line significantly inhibited AIV replication,and the deletion of ch ANP32A 125-132 AA even more significantly inhibited AIV replication.This study lays the foundation for the study of ch ANP32A gene function and the development of gene-edited chickens with resistance to avian influenza viruses and no change in the biological function of ch ANP32A.
作者
魏天悦
于萌萌
王震
谢雨杭
王晓钧
夏长友
孟庆文
WEI Tianyue;YU Mengmeng;WANG Zhen;XIE Yuhang;WANG Xiaojun;XIA Changyou;MENG Qingwen(Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,State Key Laboratory for Animal Disease Control and Prevention,Harbin 150069,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2024年第11期1437-1442,共6页
Chinese Veterinary Science
基金
国家重点研发计划项目(2022YFF0710501,2022YFF0711005)
中央级公益性科研院所基本科研业务费专项(1610302022018)。
