利用CRISPR/Cas9技术构建PRVGD株gI和gE缺失毒株及其生物学特性研究

Construction of PRV GD gI and gE Deletion Strain by CRISPR/Cas9Technique and Preliminary Study on Its Biological Characteristics

本研究利用CRISPR/Cas9基因编辑技术对本实验室分离得到的PRV GD株的gI和gE基因进行基因敲除,经蚀斑纯化结合PCR鉴定的方法获得gI和gE基因缺失毒株,命名为PRV GD-delgI/gE。试验测定了该基因缺失病毒在PK-15细胞中培养的一步生长曲线及其对小鼠的致病力。试验结果表明,与亲本毒株PRV GD株相比,PRV GD-delgI/gE在PK-15细胞中的繁殖能力有轻微降低,对小鼠的毒力明显降低,且该基因缺失病毒遗传稳定,可作为新型PRV疫苗的候选毒株,并为针对变异毒株的控制和新型疫苗的研制奠定了一定的基础。

In this study,the gI and gE genes of PRV GD strain isolated by researchers in our laboratory were knocked out by CRISPR/Cas9 gene editing technology,and the gI and gE genes deletion virus which was named as PRV GD-delgI/gE strain was obtained by plaque purification and PCR identification.We studied the one-step growth curve of PRV GD-delgI/gE strain in PK-15 cell and the virulence of the virus to mice.The results showed that PRV GD-delgI/gE strain had a slightly decreased reproductive capacity in PK-15 cells and a significantly reduced toxic to mice when compared with PRV GD strain.Moreover,PRV GD-delgI/gE strain is genetically stable and can be used as a candidate strain of new PRV vaccine,which lays the groundwork for the control of PRV variant strains and the development of new PRV vaccine.