禽传染性支气管炎病毒感染性克隆的快速构建及应用  

Rapid construction and application of infectious clone for avian infectious bronchitis virus

作　　者：卢渊录 曾亦然 罗皓炜 乔炳晨 孟琪 刘志远 王蔚怡 陈纳 赵令才 孟宪臣 张海涛 平继辉[1] LU Yuan-lu;ZENG Yi-ran;LUO Hao-wei;QIAO Bing-chen;MENG Qi;LIU Zhi-yuan;WANG Wei-yi;CHEN Na;ZHAO Ling-cai;MENG Xian-chen;ZHANG Hai-tao;PING Ji-hui(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Lihua Animal Husbandry company limited,Changzhou 213168,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏立华牧业股份有限公司,江苏常州213168

出　　处：《中国预防兽医学报》2024年第4期333-341,349,共10页Chinese Journal of Preventive Veterinary Medicine

基　　金：中央高校基本科研业务费专项资金资助(YDZX2023005)。

摘　　要：为构建高效且易于操作传染性支气管炎病毒(IBV)基因组的反向遗传学平台,本研究将IBV H120株全基因组经RT-PCR分段扩增,并在其基因组的5'端引入人巨细胞病毒(CMV)启动子序列以及3'端引入丁型肝炎病毒核酶序列(HDVRz)和牛生长激素(BGH)多聚腺苷酸化信号序列(后续称HB序列),获得H120株基因组的9个片段(CMV-F1、F2、F3、F4、F5、F6、F7、F8和F9-HB);其中片段F7通过引物引入沉默突变C20356T作为遗传标记。采用转化偶联重组(TAR)克隆技术将9个DNA片段与线性质粒p YES1L转化酿酒酵母细胞,快速构建H120株基因组全长cDNA克隆,经连接PCR(Junction-PCR)筛选及测序鉴定获得感染性克隆质粒p YES1L-H120。为了提高病毒拯救效率,同时构建表达IBV N蛋白的辅助质粒pcDNA3.1-N+3'UTR。将p YES1L-H120与pcDNA3.1-N+3'UTR共转染BHK-21细胞,48 h后收获细胞,接种9日龄SPF鸡胚盲传3代,经胚体病变观察和测序显示获得IBV反向遗传重组病毒r H120株。为了以IBV为载体表达外源基因,本研究将病毒基因组的5a基因替换为eGFP基因,采用TAR克隆技术构建以IBV为载体表达外源e GFP基因的cDNA克隆pYES1L-H120-Δ5a-eGFP,与辅助质粒共转染BHK-21细胞,随后接种SPF鸡胚拯救嵌合病毒r H120-Δ5a-eGFP,并采用PCR和测序鉴定该重组病毒正确构建。结果显示,采用TAR克隆技术能够在短时间内获得IBV反向遗传重组病毒且快速操作病毒基因组获得以IBV为载体稳定表达外源基因的嵌合病毒。本研究首次建立了基于TAR克隆技术的IBV反向遗传操作平台,为研究病毒的生物学特性、载体疫苗的开发和抗病毒药物的筛选提供高效便捷的工具。To construct an efficient and easily manipulated reverse genetics platform for the infectious bronchitis virus(IBV)genome,we employed transformation-associated recombination(TAR)technology to rapidly construct infectious clones in Saccharomyces cerevisiae.Firstly,the genome of IBV H120 strain was amplified using RT-PCR for 9 fragments,and the human cytomegalovirus(CMV)promoter sequence was introduced at the 5'end of the genome,while the hepatitis D virus ribozyme sequence(HDVRz)and bovine growth hormone(BGH)polyadenylation signal sequence(subsequently called HB sequence)were introduced at the 3'end.A silent mutation,C20356T,was introduced into fragment F7 by primer design as a genetic marker.Subsequently,full-length cDNA clones of the H120 genome were rapidly constructed by transforming 9 DNA fragments and linear plasmid pYES1L into Saccharomyces cerevisiae cells using the TAR cloning technique.The recombinant plasmid pYES1L-H120 was obtained by Junction-PCR screening and sequencing.To improve the efficiency of virus rescue,a helper plasmid expressing the N gene of IBV,pcDNA3.1-N+3'UTR,was also constructed.BHK-21 cells were co-transfected with pYES1L-H120 and the helper plasmid pcDNA3.1-N+3'UTR.The cells were harvested 48 hours post transfection,then inoculated into 9-day-old SPF chicken embryos and passed through 3 generations.The results of somatic lesions and sequencing showed that the rH120 strain of IBV reverse genetic virus was successfully obtained.Additionally,in order to express exogenous genes using IBV as vector,we used the TAR cloning technique to construct a cDNA clone expressing the exogenous eGFP gene by replacing the 5a gene of the viral genome with the eGFP gene,and the chimeric virus rH120-Δ5a-eGFP was successfully rescued.The results showed that IBV reverse genetic recombination virus could be obtained in a short time by using TAR cloning technique.The viral genome could also be manipulated quickly to obtain the chimeric virus with stable expression of exogenous genes while using IBV as the ve

关 键 词：传染性支气管炎病毒 反向遗传学 酿酒酵母 转化偶联重组技术 外源基因 

分 类 号：S852.65[农业科学—基础兽医学]