重组禽腺联病毒传递的miRNAs对鸡传染性法氏囊病毒复制的抑制

Inhibition of infectious bursal disease virus replication in DF-1 cells by miRNAs delivered by recombinant avian adeno-associated viral vector

为了研究重组禽腺联病毒介导的基因特异miRNAs对传染性法氏囊病毒(IBDV)复制的抑制作用。在前期研究基础上,利用重组DNA技术构建表达vp2与vp1基因特异miRNAs的重组禽腺联病毒转移载体,磷酸钙法制备重组禽腺联病毒;纯化后的病毒经过电镜观察、病毒感染性试验确定其存在与感染滴度,提取转导重组病毒后的细胞总RNA,RT-PCR法检测miRNAs表达情况,在重组病毒转导的细胞中感染不同源的IBDV,在感染后不同时间测定病毒TCID50及vp2基因表达水平。结果显示,成功制备了重组禽腺联病毒,病毒感染性滴度可达5×108TU/ml,与阴性对照相比,成功表达的miRNA能在细胞上抑制病毒基因的扩增,大幅度降低同源或异源病毒的滴度。表明重组禽腺联病毒可以作为有效传送和稳定表达miRNA的载体,有望开发预防鸡传染性法氏囊病(IBD)的新方法。

To study the inhibition of infectious bursal disease virus（IBDV） replication by miRNAs delivered by re-combinant avian adeno-associated viral vector, the recombinant avian adeno-associated virus transfer vector for vp2 or vp1gene-specific miRNAs was built by recombinant DNA technology. Recombinant avian adeno-associated virus was prepared bycalcium phosphate method. Virus infestion test revealed that the physical titre reached 5×10^8 TU/ ml of purified virus. The to-tal cellular RNA was extracted after the transduction of recombinant virus for detection of expressed miRNAs. After transduc-tion for two days, the DF-1 cells were infected with homologous or heterologous IBDV. Then vp2 gene expression level and vi-rus TCID50 were tested. Compared with the negative control group, the successful expression of miRNA could significantly in-hibit gene amplification on homologous or heterologous viruswhich was showed by declined titres. Recombinant avianadeno-associated virus could be applied as an effectivelyand stably expressing vector of miRNA used in develop anew method to prevent infectious bursal disease（IBD）.