Generation of recombinant lentogenic Newcastle disease virus from cDNA
Generation of recombinant lentogenic Newcastle disease virus from cDNA
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DOI：
10.1099/0022-1317-80-11-2987
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发表时间：
1999-11-01
影响因子：
3.8
通讯作者：
Mettenleiter, TC
中科院分区：
文献类型：
--
作者：
Römer-Oberdörfer, A;Mundt, E;Mettenleiter, TC
将抗基因组意义的新城疫病毒RNA和新城疫核蛋白、磷酸化蛋白和依赖RNA的RNA聚合酶同时转染到稳定表达T7 RNA聚合酶的细胞中，克隆-30疫苗株可重复产生重组新城疫病毒（NDV）。为此，在T7 RNA聚合酶启动子的控制下，克隆了包含15186 nt的clone -30基因组，并对其进行了测序，然后组装成全长cDNA克隆。将转染上清液接种于SPF鸡胚尿囊腔中扩增重组病毒。在子代病毒中存在人工引入非编码区共包含5个核苷酸变化的两个标记限制性位点。重组新城疫病毒在细胞培养和胚卵中的生长特征与亲本野生型病毒没有明显区别。通过对SPF日龄鸡进行脑内接种，两种病毒的脑内致病性指数均为0.29，证明重组NDV是NDV亲本疫苗株的忠实拷贝。
Recombinant lentogenic Newcastle disease virus (NDV) of the vaccine strain Clone-30 was reproducibly generated after simultaneous expression of antigenome-sense NDV RNA and NDV nucleoprotein, phosphoprotein and RNA-dependent RNA polymerase from plasmids transfected into cells stably expressing T7 RNA polymerase. For this purpose, the genome of Clone-30, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. Two marker restriction sites comprising a total of five nucleotide changes artificially introduced into noncoding regions were present in the progeny virus. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in cell culture and in embryonated eggs. Moreover, an intracerebral pathogenicity index of 0.29 was obtained for both viruses as determined by intracerebral inoculation of day-old SPF chickens, proving that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV.