摘要
EP0基因是伪狂犬病毒(Pseudorabiesvirus,PRV)的早期基因,可能与病毒复制及潜伏感染等有关。为了筛选特异性抑制EP0基因表达的siRNA序列,本研究按照Ambion公司公布的siRNA分子设计原则,设计并合成了3个针对PRVEa株EP0基因的siRNA模板EP04、EP08和EP12,分别克隆到以CMV启动子的siRNA表达载体pSilencer4.1-CMVneo中,构建相应的重组表达质粒p4.1-EP04、p4.1-EP08和p4.1-EP12。同时,将EP0基因的编码区克隆到真核表达载体pEGFP-N3中,按照读码框架与EGFP基因的5’端融合,获得重组表达质粒pEP0-EGFP。将p4.1-EP04、p4.1-EP08、p4.1-EP12和阴性对照质粒p4.1-NK分别与pEP0-EGFP共转染IBRS-2细胞,荧光显微镜观察、流式细胞仪和半定量RT-PCR检测结果表明,三个siRNA分子均能不同程度地抑制EP0基因的表达,抑制效率从高到低依次为EP08、EP12和EP04;在进一步的病毒感染实验中也得到了与细胞转染模型一致的结论。这为深入研究EP0基因在PRV复制和潜伏感染中的作用奠定了基础。
EP0, an early protein gene of pseudorabies virus （PRV）, plays important roles in viral replication and possibly in PRV latency. In order to select an siRNA that specifically inhibits the expression of EP0, three siRNA templates, EP04, EP08 and EP12, were designed and synthesized according to the EP0 sequence of PRV Ea strain and the siRNA design guidance of Ambion, and then cloned into the siRNA expression vector pSilencer 4.1-CMV neo containing a CMV promoter. This resulted in three recombinant plasmids p4.1-EP04, p4.1-EP08 and p4.1-EP12. Another recombinant plasmid pEP0-EGFP was also constructed by an in-frame fusing the EP0 coding sequence to the 5＇-end of the EGFP gene in the expression vector pEGFP-N3. The p4.1-EP04, p4.1-EP08, p4.1-EP12 and a negative control p4.1-NK were separately co-transfected into IBRS-2 cells. Fluorescence microscopic observation, flow cytometric and RT-PCR analysis revealed that all the three siRNA could inhibit EP0 expression in the model as well as in PRV replication, with EP08 being the most efficient one, followed by EP12 and EP04. Our data are important to further study the function of EP0 in PRV replication and latency.
出处
《中国病毒学》
CSCD
2006年第6期565-570,共6页
Virologica Sinica
基金
国家自然科学基金(30471293)
