鸭肠炎病毒疫苗株UL41和UL40基因间隔区作为其复制非必需区的鉴定

Identification of spacer regions of UL41 and UL40 genes of duck enteritis virus vaccine strains as non-essential regions for replication

为探寻鸭肠炎病毒(DEV)基因组中可插入外源基因的复制非必需区,本研究利用CRISPR/Cas9基因编辑技术,构建重组质粒pX459-ul4140和含有红色荧光蛋白(RFP)表达框架的穿梭片段UL41-RFP-UL40,转染后拯救重组病毒,并对其进行噬斑纯化。通过PCR和荧光显微镜观察对重组病毒进行鉴定,结果显示,经拯救得到表达RFP的重组病毒rDEV-ul4140RFP;所有代次的rDEV-ul4140RFP在显微镜下均能观察到红色荧光,表明重组病毒中的RFP基因稳定存在。将rDEV-ul4140RFP和亲本病毒DEV疫苗株接种鸡胚成纤维细胞(CEFs)后,收集24 h、48 h、72 h、96 h的细胞和上清,检测TCID并绘制生长曲线。结果显示,rDEV-ul4140RFP在CEF中的生长曲线和亲本DEV疫苗株无显著差异。利用SPF鸭对rDEV-ul4140RFP的免疫保护效果进行评价。攻毒保护实验结果显示,rDEV-ul4140RFP和亲本病毒DEV疫苗株均能对SPF鸭提供100%的免疫保护。本研究结果证实DEV基因组UL41和UL40基因间隔区可作为外源基因的插入位点,为以DEV为载体构建相关重组疫苗提供参考依据。

To explore the viral replication non-essential regions of duck enteritis virus(DEV) in which exogenous genes can be inserted, the recombinant plasmid pX459-ul4140 and the shuttle fragment UL41-RFP-UL40 containing red fluorescent protein(RFP) expression framework were constructed by CRISPR/Cas9 gene editing technology. The recombinant virus rDEV-ul4140RFP was rescued and purified. The obtained recombinant virus was identified by PCR and fluorescence analysis. The results showed that the recombinant plasmid and shuttle fragment were successfully constructed and rDEV-ul4140RFP expressing red fluorescent protein was obtained. RFP gene stably existed in all generations of rDEV-ul4140RFP and red fluorescence could be observed under microscope. After the inoculation of chicken embryo fibroblasts(CEFs) with rDEV-ul4140RFP or the parental DEV vaccine strain,the cells and supernatants were collected for 24 hours, 48 hours, 72 hours and 96 hours, and the TCIDwas detected and the growth curve was drawn. The results showed that rDEV-ul4140RFP had proliferation efficiency similar to the DEV vaccine strain on CEFs.The protective efficacy of the recombinant virus against DEV was evaluated in SPF ducks. Results demonstrated that both of them can provide complete protection against DEV. In this study, UL41 and UL40 gene spacer regions of DEV genome were identified as insertion sites of foreign genes, which provides a basis for the construction of related recombinant vaccine with DEV.