鸭瘟病毒疫苗株基因组复制非必需区的筛选

Selection of nonessential regions for viral replication of duck enteritis virus(vaccine strain) genome

为探寻鸭瘟病毒(DEV)基因组中的复制非必需区,本研究在已建立的DEV疫苗株5粘粒拯救系统的基础上,利用转座子随机插入技术,将含eGFP表达框架的基因片段随机插入DEV US区,救获两株表达绿色荧光蛋白的重组DEV(rDEV-us7eGFP、rDEV-us8us1eGFP)。将两株重组病毒在鸡胚成纤维细胞中连续传至20代后,通过PCR、荧光表达鉴定分析,结果显示gfp基因能够在重组病毒中稳定存在并正确表达;生长动力学结果表明,重组病毒在CEF中的增殖效率与DEV疫苗株无显著差异。攻毒保护实验结果表明,将rDEV-us7eGFP和rDEV-us8us1eGFP以10~5TCID_(50)的剂量免疫SPF鸭后,其对鸭瘟强毒攻击的保护效率与DEV疫苗株一致,均为100%。本研究成功筛选到DEV基因组中的两个可供外源基因插入的复制非必需区,分别位于us7基因内部及us8与us1基因之间,为以DEV为载体构建相关重组疫苗奠定了重要基础。

To identify nonessential regions for viral replication of duck enteritis virus （DEV） genome, five recombinant fosmids covering the whole genome of DEV developed in our laboratory were used in this study. The genome fragment containing eGFP gene were inserted into the unique long （UL） region randomly by use of transposons, and the two recombinant viruses expressing green fluorescent protein were successfully rescued. Both of recombinant viruses were serially passed in CEF cells for 20 passages. The identified results indicated that GFP gene was stably existed in both of the recombinant viruses detected by PCR, and the in vitro tests showed that the inserted gene was able to express stably without affecting the recombinant virus replication in cells. The results of virulence and immunogenicity evaluation of the two recombinant viruses indicated that the viruses maintained replication and immunogenic properties, which were similar to those of the parent DEV strain. In conclusion, we found another two nonessential regions sites, one was located inside us7 gene and another was between us8 and us1 gene for the exogenous gene insertion and expression, which provided a basis for development of the recombinant live attenuated DEV vaccines.