通过反向遗传操作技术构建传染性支气管炎病毒nsp15核酸内切酶活性缺陷型重组毒株  

Construction of Infectious Bronchitis Virsus Endoribonucleasensp 15 Defective Recombinant Virus by Reverse Genetic System

作　　者：翁文莲 宫晓倩[1] 高波 方守国[2] 王欢 钱琨 丁铲[1,3] 廖瑛 WENG Wenlian;GONG Xiaoqian;GAO Bo;FANG Shouguo;WANG Huan;QIAN Kun;DING Chan;LIAO Ying(Department of Avian Diseases,Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai,200241,China;College of Agriculture,College of Animal Sciences,Yangtze University,Jingzhou 434025,China;Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou,225009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]长江大学农学院/动物科学院,荆州434000 [3]江苏省人兽共患病学重点实验室/江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州大学,扬州225009

出　　处：《病毒学报》2021年第6期1376-1384,共9页Chinese Journal of Virology

基　　金：国家自然科学基金资助项目(项目号:31772724),题目:应激颗粒作为天然免疫信号枢纽的研究;中央公益性科研机构基础研究基金(项目号:2019JB03),题目:传染性支气管炎病毒延迟干扰素反应的机制研究;江苏省人兽共患病学重点实验室资助项目(项目号:R2007),题目:冠状病毒拮抗干扰素反应的研究。

摘　　要：传染性支气管炎病毒(Infectious bronchitis virus,IBV)属于γ属冠状病毒,其核酸内切酶nsp15(Non-structural protein 15)的功能尚未得到解析。为探讨nsp15在IBV感染和复制过程中的作用,本研究对nsp15的核酸内切酶活性中心H238进行突变,通过体外连接技术构建重组病毒rIBV-nsp15-H238A,并对病毒滴度、感染复制能力和生长曲线进行鉴定。方法如下:将IBV Beaudette-R毒株的基因组分成5个片段克隆到质粒载体,并在每个片段加上Bsa I或Bsm BI限制性酶切位点。通过质粒载体进行扩增后,通过酶切获得cDNA片段,在体外连接成全长基因组cDNA,转录出全长基因组RNA,跟N的转录本一起电击转染到Vero细胞,拯救出重组病毒rIBV和rIBV-nsp15-H238A。利用空斑试验检测比较rIBV和rIBV-nsp15-H238A的病毒滴度和空斑大小,鉴定nsp15核酸内切酶活性对IBV感染性的影响。结果显示,rIBV-nsp15-H238A的病毒滴度为2.7×10^(6)PFU/mL,比rIBV的病毒滴度(9.4×10^(6)PFU/mL)低3倍还多,且rIBV-nsp15-H238A空斑孔径远小于rIBV,说明rIBV-nsp15-H238A复制和扩散能力远低于rIBV。利用TCID_(50)检测病毒的生长曲线,结果表明在感染后0~24h,rIBV-nsp15-H238A的生长曲线均低于rIBV。由此可见,IBV nsp15H238核酸内切酶活性位点对IBV的感染复制起着重要作用。本研究通过反向遗传操作构建nsp15核酸内切酶活性缺陷型重组病毒,为研究nsp15的功能提供了一个有力的工具。Infectious Bronchitis Virus(IBV) belongs to the γ coronavirus,however,the function of IBV encoded endoribonuclease(non - structural protein 15,nsp15)has not been determined yet. To explore the function of nsp15 in the process of IBV replication,we mutated the IBV nsp15 endonuclease core residue His238 to Ala,constructed the nsp15-defective recombinant virus rIBV-nsp15-H238 A,via in vitro ligation and recombination technology. Plaque assay and TCID_(50)were performed to measure virus titer,virus plaque size and growth curve.The IBV Beaudette-R genome was cloned as 5 fragments in vectors,Bsa I or Bsm BI restriction sites were added to the end of each fragment. After plasmid amplification,the cDNA fragments were obtained by enzymatic digestion,followed with in vitro ligation and transcription. Full - length genomic RNA was electroporated into Vero cells,together with N transcript,to rescue the recombinant viruses rIBV and rIBV -nsp15 - H238 A. Plaque assay was performed to detect and compare the viral titer and plaque size of these two recombinant viruses. Results showed that the virus titer of rIBV - nsp15 - H238 A was 2.71×10^(6) PFU/mL,3 times lower than that of rIBV(9.4×10^(6) PFU/mL). The plaque size of rIBV-nsp15-H238 A was much smaller than that of rIBV,indicating that rIBV - nsp15 - H238 A replicates and spreads slower than rIBV. The growth curve of rIBV-nsp15-H238 A was slower than that of rIBV. Our study demonstrates that nsp15 H238 is the key amino acid and plays an important role in the replication and spread of IBV. The construction of nsp15 defective recombinant virus provides a powerful tool for the study of the function of nsp15.

关 键 词：传染性支气管炎病毒(IBV) 体外连接 感染性cDNA克隆 rIBV rIBV-nsp15-H238A 

分 类 号：S855[农业科学—临床兽医学]