1型鸭肝炎病毒CL株感染性分子克隆的构建

Construction of infectious molecular clone of Duck hepatitis virus type 1 CL strain

摘要:

摘要：【目的】构建1型鸭肝炎病毒（DHV）的感染性克隆，用于研究其基因组的结构与功能。【方法】用RT-PCR方法扩增出覆盖整个1型鸭肝炎病毒CL株基因组3个忠实性片段，并按顺序组装进载体pBR322中，获得全长cDNA克隆（BR-CL）。将BR-CL在体外转录出的RNA转染鸭胚肾细胞，并传至第6代，利用RT-PCR方法和间接免疫荧光试验进行鉴定。将获得的子代病毒（CL-R）在SPF鸡胚上传代，观察鸡胚死亡及胚体病变情况。通过胶体金免疫电镜观察子代病毒粒子的形态。【结果】RT-PCR、间接免疫荧光和胶体金免

Abstract:

Abstract: [Objective] To construct an infectious clone for studying functions of duck hepatitis virus(DHV) type1 genome by reverse genetic technique. [Methods] Three fidelity DNA fragments covering the full genome of DHV type1 CL strain were amplified by RT-PCR, and inserted into pBR322 vector, resulting in the full-length cDNA clone BR-CL. The in vitro-transcribed RNA from BR-CL was transfected into duck embryo renal cells and the rescued virus was identified using RT-PCR, indirect immunofluorescence assay and colloidal gold immunoelectron microscopy after six generations. After inoculating the rescued virus into SPF chick embryos, embryo death and pathological changes were observed. [Results] The results of RT-PCR, indirect immunofluorescence assay and immunoelectron microscope showed that infectious virus was rescued. After inoculating into SPF chick embryos, the rescued virus was able to kill embryos with pathogenic changes. [Conclusion] This is the first report on generation of infectious cDNA clone of DHV, which provides a valuable platform for further research on functions of DHV genome.