基于单质粒拯救系统的塞内卡病毒感染性克隆的构建与生物学特性

Construction and biological characterization of an infectious clone of Senecavirus A based on a single plasmid rescue system

参考甲型塞内卡病毒(SVA)CH/HuB/2017株全基因组序列,构建SVA通用型载体,合成SVA FragⅠ和FragⅡ基因片段,并通过无缝克隆技术依次将病毒基因片段插入通用型载体,获得包含病毒全基因组的真核转录质粒pcDNA-SVA;经双酶切和测序验证后,在IBRS-2细胞上进行转染和传代,以获得拯救毒株rSVA-CH/HuB/2017;经遗传稳定性评价后,利用蛋白免疫印迹、间接免疫荧光和半数组织培养感染量等试验,鉴定和分析拯救毒株在猪肾细胞系IBRS-2和PK-15中的生物学特性。结果显示,重组质粒pcDNASVA直接转染普通敏感细胞系IBRS-2,即可快速拯救出含有沉默突变NheⅠ分子标记的毒株,且具有良好的遗传稳定性;拯救毒株和野生亲本毒株在IBRS-2和PK-15细胞中的复制和增殖特性相似。结果表明,通过建立一种基于T7启动子、RNA聚合酶Ⅱ启动子、终止子、核酶HamRz和HDVRz等元件的SVA单质粒高效拯救系统,可以在质粒水平快速编辑病毒基因组,为定向改造和拯救病毒提供了基础性技术平台。

Senecavirus A(SVA)universal vector was constructed and SVA FragⅠand FragⅡgene fragments were synthesized according to the whole genome sequence of CH/HuB/2017 strain.Then,SVA FragⅠand FragⅡwere inserted into the SVA universal vector successively by seamless cloning technique to obtain the eukaryotic transcription plasmid pcDNA-SVA containing the whole genome of the virus.The constructed plasmid was validated by double digestion and sequencing,and then was transfected into IBRS-2 cells to obtain the rescue strain rSVA-CH/HuB/2017.After the genetic stability evaluation,the biological characteristics of rescue virus in porcine kidney cell lines IBRS-2 and PK-15 were identified and analyzed by Western-blot,indirect immunofluorescence assay and TCID50.The results showed that the recombinant plasmid pcDNA-SVA could be directly transfected into common sensitive cell line IBRS-2,which could quickly rescue the virus containing the silent mutant NheⅠmolecular marker with good genetic stability.The replication and proliferation characteristics of the rescue strain and wild-type parent strain were similar in IBRS-2 and PK-15 cells.By establishing an efficient single-plasmid rescue system of SVA based on T7 promoter,RNA polymeraseⅡpromoter,terminator,ribozyme HamRz and HDVRz,the virus genome can be rapidly edited at the plasmid level,providing a technical platform for targeted modification and rescue of the virus.