犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

Rescue of lesser panda canine distemper virus by reverse genetic manipulation

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬瘟热病毒感染性cDNA克隆。对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒。酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础。

In this study,the CDV originated from lesser pandas was isolated and served as the template for the infectious cDNA clone of CDV.After genome sequencing,seven cDNA fragments containing full-length CDV genome sequence were obtained by RT-PCR.The seven different fragments were digested and spliced together,and then inserted into eukaryotic expression vector pCI.Thus,we obtained the full-length cDNA of CDV lesser panda named pCI-CDV-LP.Furthermore,three helper plasmids were constructed by cloning the N,P,L protein ORF of lesser panda strain CDV in pCI vectors,respectively.The sequences of nuclease and CDV cDNA in pCI vector were verified by restriction enzyme digestion and sequencing.The full-length plasmid and three helper plasmids were then co-transfected into BSR cells using transfection reagent Lipofectamine TM2000.The results of RT-PCR,indirect immunofluorescence assay and viral infection assay showed that the lesser panda CDV was rescued successfully,and the reverse genetics of lesser panda CDV was built successfully.The results of the current investigation provide an important insight into the pathogenesis of canine distemper virus.