犬瘟热病毒CDV-3株感染性克隆的构建与鉴定

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus

摘要:

以国内商品化水貂犬瘟热病毒疫苗所用毒株CDV-3为模板，构建犬瘟热病毒感染性cDNA克隆，为犬瘟热病毒新型疫苗研制、致病机理研究提供理论基础。设计13对引物对其全基因组序列测定，分析单一酶切位点，将CDV-3的全长分5个片段进行RT-PCR扩增。经酶切拼接，将5个片段顺次插入到酶切位点改造后的真核载体pcDNA3.2的多克隆酶切位点处，同时在F1首端和F5末端分别加入锤头状核酶和丁型肝炎核酶序列，获得CDV-3株的全长cDNA质粒 (pcDNA3.2-CDV-3)。构建表达CDV-3 N、P、L蛋白的3个辅助质粒。利用转染试剂LipofectamineTM 2000将全长质粒和3个辅助质粒共转染293T细胞，3 d后，将上清接种到Vero细胞。观察犬瘟热病毒典型合胞体病变，对重组病毒进行免疫荧光鉴定和标签鉴定。最后，比较wtCDV-3和rCDV-3的生长特性。全长质粒和辅助质粒的酶切鉴定和序列测序均正确。拯救的重组病毒能在Vero细胞上形成典型的合胞体病变，经RT-PCR、间接免疫荧光和电镜观察鉴定，证明成功拯救出重组病毒rCDV-3株。rCDV-3的病毒滴度最高达到107.667 TCID50/mL，比wtCDV-3的滴度106.667 TCID50/mL高出约10倍。rCDV-3感染Vero细胞后，迅速大量增殖，于感染后36 h达到最高病毒滴度。而wtCDV-3增殖平缓，感染后72 h时病毒含量达到最大。文中建立的高效CDV-3株反向遗传操作平台，为犬瘟热病毒新型疫苗研制和致病机理研究奠定基础。

Abstract:

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by LipofectamineTM 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 107.667 TCID50/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 106.667 TCID50/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.