基于CRISPR/Cas9的羊口疮病毒dUTPase基因缺失株构建及其生物学特性

Orf, also known as contagious ecthyma, is an contagious disease caused by orf virus (ORFV). The disease has an economic impact on sheep industry due to decreases in production. The previous study has found that dUTPase gene of ORFV encoded a protein with biological activity. However, the biological function is unclear.There is a hypothesis that the dUTPase may be ORFV's virulence gene based on gene homologous compared with other viruses. However, the opinion lacks experimental basis and remains disputes. In the present project, CRISPR/Cas9 system will be employed for the dUTPase gene deletion. The sgRNA oligos were designed and cloned to PX459 vector. Then, the homology arms of dUTPase gene and RFP expression cassette were cloned to the pcDNA3.1 plasmid. Moreover, the two recombinant plasmids above will be co-transfected into MDBK cells with ORFV genomic DNA. After been fluorescent screening, plaque purification and PCR, Western blot and IFA identification, the dUTPase gene deletion mutant was obtained. Furthermore, the growth properties, pathogenicity and immunogenicity of the dUTPase gene deletion mutant was comprehensively studied at the cellular level and animal experiments. The project will clarify whether the dUTPase gene is a virulence gene of ORFV and the influence of the gene deletion on the biological feature and immunogenicity. In conclusion, this project will lay the foundation for revealing the pathogenesis of ORFV and vaccine development of the gene deletion mutant .

羊口疮又名羊传染性脓疱，是由羊口疮病毒(Orf virus,ORFV)引起的接触性传染病，严重危害养羊业的发展。前人研究发现ORFV的dUTPase蛋白具有生物学活性，但其生物学功能尚不清楚。有人根据病毒基因同源性提出其可能是ORFV毒力基因的假说，但缺乏实验依据，尚存争议。本项目拟利用CRISPR/Cas9系统，设计sgRNA oligos并克隆至PX459载体，将其与含同源臂及RFP表达盒的重组质粒，以及ORFV DNA共转染MDBK细胞。经荧光筛选、噬斑纯化、PCR、Western blot及IFA鉴定获得dUTPase基因缺失株。再从细胞水平和动物实验对基因缺失株的生长特性、毒力及免疫原性进行全面的研究。本项目有望通过实验明确dUTPase基因是否为ORFV的毒力基因，阐明dUTPase基因缺失对其生物学特性及免疫原性的影响。为揭示ORFV致病机理及基因缺失活疫苗研究奠定基础。

羊口疮又名羊传染性脓疱，是由羊口疮病毒(Orf virus,ORFV)引起的接触性传染病，严重危害养羊业的发展。本项目主要开展了下列工作：(1)利用CRISPR/Cas9系统成功构建了一株ORFV dTUPase基因缺失株rORFV△dUTPase。(2) 比较了rORFV△dUTPase与ORFV亲本毒株在细胞上的生长特性及毒力，包括一步生长曲线和遗传稳定性。(3) 研究比较了rORFV△dUTPase与ORFV亲本毒株对实验兔和山羊的毒力，包括黏膜损伤程度及组织病理学变化差别。(4) 研究比较了rORFV△dUTPase与ORFV亲本毒株感染山羊后引发的天然免疫应答，探讨dUTPase基因缺失对ORFV免疫原性的影响。包括诱导I型IFN在体内表达的情况，STAT3的激活情况以及抗病毒蛋白基因ISG15、ISG20和IFITM1表达的影响，诱导宿主炎症细胞因子(IL-1β、IL-6、IL-8和TNF-α)表达的影响，以及对TLRs在体内表达和诱导STING 信号通路关键分子的表达的影响。结果表明，dUTPase基因的缺失，对ORFV亲本毒株在OFTu细胞上的生长特性和毒力没有显著影响。兔子和山羊的实验结果表明，dUTPase基因的缺失，不会造成对实验兔唇部和耳部的损伤程度出现明显差异，不会对山羊的口唇损伤程度造成显著差异。最后，通过对山羊感染dUTPase基因缺失株和亲本毒株产生的天然免疫反应来看，dUTPase基因的缺失不会显著影响口疮病毒诱导宿主天然免疫的水平。(5) 对ORFV的dUTPase、020、024、112、127、VEGF多个基因进行了克隆、原核表达及多抗血清的制备或亚细胞定位。对ORFV的023和VIR基因进行了克隆及生物信息学分析。(6) 建立了ORFV的SYBR Green荧光定量PCR检测方法。(7) 构建并鉴定了稳定表达ORFV B2L基因的重组腺病毒。. 通过本项目的实施，为日后深入研究ORFV dUTPase基因和其他多个病毒蛋白的功能奠定了一定的基础，提供了宝贵的生物材料。为临床快速、准确诊断ORFV提供了检测方法，也为ORFV疫苗的研制奠定了一定的基础。