塞内卡病毒CH/ZZ/2016株cDNA感染性克隆的构建  

Construction of Infectious cDNA Clone of Senecavirus CH/ZZ/2016

作　　者：韩宇 王秀明 刘建奇 刘亭岐 乌吉斯古楞 张斌 宋庆庆 关平原[1] HAN Yu;WANG Xiuming;LIU Jianqi;LIU Tingqi;Wujisiguleng;ZHANG Bin;SONG Qingqing;GUAN Pingyuan(Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases Ministry of Agriculture and Rural Affairs,College of Veterinary Medicine Inner Mongolia Agricultural University,Hohhot 010018,China;JINYUBAOLING Bio-Pharmaceutical Co.,Ltd,National Engineering Laboratory,Hohhot 010020,China)

机构地区：[1]内蒙古农业大学兽医学院农业农村部动物疾病临床诊疗技术重点实验室,呼和浩特010018 [2]金宇保灵生物药品有限公司兽医疫苗国家工程实验室,呼和浩特010020

出　　处：《中国动物传染病学报》2023年第2期67-72,共6页Chinese Journal of Animal Infectious Diseases

摘　　要：为构建塞内卡病毒(Senecavirus A)CH/ZZ/2016株的感染性克隆,采用RT-PCR技术分3个cDNA片段对SVA/CH/ZZ/2016株全基因组进行扩增,并克隆至pcDNA3.1(+)真核表达质粒中。获得重组质粒pSVA-CH/ZZ经NheⅠ、KpnⅠ酶切及测序鉴定后,转染BHK-21细胞并转至PK-15细胞上进行盲传,直至出现细胞病变(CPE)。对出现CPE的细胞上清液进行RT-PCR扩增、酶切、测序、间接免疫荧光试验鉴定。结果显示,成功构建了含SVA全长cDNA克隆的重组质粒,转染BHK-21细胞经PK-15细胞盲传至F5代出现SVA的典型CPE。细胞上清液经RT-PCR扩增、酶切、测序结果表明拯救病毒含有不同于亲本病毒的分子标记。间接免疫荧光试验进一步表明拯救了SVA病毒。病毒蚀斑形成和生长曲线表明,拯救毒株和亲本毒株的复制能力及增殖特性相似。本研究构建的CH/ZZ/2016株感染性克隆为进一步研究SVA的致病机理、基因功能及疫苗的研发奠定基础。To develop the infectious cDNA clone of Senecavirus A strain CH/ZZ/2016,its whole genome was amplified by RT-PCR technology into 3 cDNA fragments,which were then cloned into the pcDNA3.1(+)plasmid.The obtained recombinant plasmid pSVA-CH/ZZ was digested with Nhe I and Kpn I and identified by sequencing,followed by transfection into BHK-21 cells.The cell supernatant was collected and transferred to PK-15 cells for blind passages till the fifth passage with SVA typical CPE.The cell supernatant with CPE was identified by RT-PCR amplification,restriction enzyme digestion,sequencing,and indirect immunofluorescence test.The identification by restriction enzyme digestion and sequencing showed that the recombinant plasmid containing the full-length cDNA clone of SVA was successfully constructed,which was then transfected into BHK-21 cells developing typical SVA CPE at the fifth passage.The successful rescue of rSVA virus was confirmed by amplification of cell supernatant by RT-PCR,enzyme digestion,sequencing,and indirect immunofl uorescence test identification.The Plaque phenotype and virus growth curve showed that the rSVA and parental strain were basically identical in terms of replication ability and in vitro growth ability.The infectious clone of CH/ZZ/2016 strain constructed in this study laid the foundation for further research on the pathogenic mechanism,gene function and vaccine development of SVA.

关 键 词：塞内卡病毒 全长CDNA 转染 病毒拯救 

分 类 号：S852.65[农业科学—基础兽医学]