靶向S基因siRNA抑制猪血凝性脑脊髓炎病毒在N2a细胞中的复制  

Inhibition of porcine hemagglutinating encephalomyelitis virus(HEV) replication by siRNA targeting the spike glycoprotein(S) gene in N2a cells

作　　者：兰云刚[1] 赵魁[1] 王改丽[1] 李明谦[1] 贺文琦[1] 陆慧君[2] 陈克研[1] 岳占碰[1] 高丰[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林大学人畜共患病研究所人畜共患病教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2011年第3期303-308,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30871849;31072134);吉林省科技发展国际合作项目(20080722);吉林省青年科研基金资助项目(20090154)

摘　　要：以猪血凝性脑脊髓炎病毒(HEV)的S糖蛋白基因作为靶基因,设计并体外转录合成2对小干扰RNA(siRNA),分别为SS1和SS2。将SS1和SS2分别转染小鼠成神经瘤细胞(N2a)并在转染后接种HEV,48h后通过间接免疫荧光、血凝试验、TCID_(50)测定以及荧光定量PCR等方法对SS1和SS2抑制HEV在N2a细胞中增殖的效果进行了研究。结果显示,转染细胞感染HEV48 h后,SS1和SS2干扰组细胞出现的绿色荧光明显少于对照组,血凝效价分别是对照组的1/18和1/32,病毒感染滴度分别是对照组的1/28和1/103,两干扰组的HEV基因组的拷贝数分别是对照组的76%和84%。以上结果表明,SS1和SS2均对HEV在N2a细胞中的复制具有不同程度的抑制作用,其中以SS2的抑制作用最为显著。该项试验研究结果不仅为HEV致病机制的研究奠定基础,同时可为抗HEV新型药物及抗病育种新靶点的设计奠定分子生物学基础。The specific effect of RNAi on the replication of HEV was explored.Two species of small interfering RNA （siRNA） named SSI and SS2,targeting different regions of the HEV spike glycoprotein（S） gene,were prepared by in vitro transcription.After transfection of N2a cells with each of the siRNAs followed by infection with HEV.At 48 hours post-infection,infected cells were evaluated for antiviral activity against the HEV strain by indirect immuno-fluorescence microscopy（IFA）.hemagglutination（HA） titre,virus titration and real-time RT-PCR.At 48 hours post-infection,only a few siRNA-treated cells were positive for viral antigen staining,whereas most untreated virus-infected cells were positive.Transfection with siRNAs also suppressed the production of infectious virus up to 18-32- fold as assessed by a hemagglutination（HA） test and 28-103-fold as assessed by TCID50 assay.Furthermore,treatment with siRNAs caused a 76%-84%reduction in viral genome copy number as assessed by real time RT-PCR. These results suggested that the two species of siRNAs can efficiently inhibit HEV genome replication and infectious virus production especially SS2.In future studies,a combination of siRNAs targeting the S gene may be used as a tool to study HEV replication,antiviral therapy and transgenic animals.

关 键 词：S基因 SIRNA 复制 

分 类 号：S852.65[农业科学—基础兽医学]