Heterologous gene expression from transmissible gastroenteritis virus replicon particles
Heterologous gene expression from transmissible gastroenteritis virus replicon particles
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DOI：
10.1128/jvi.76.3.1422-1434.2002
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发表时间：
2002-02-01
影响因子：
5.4
通讯作者：
Baric, RS
中科院分区：
文献类型：
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作者：
Curtis, KM;Yount, B;Baric, RS
我们最近分离到一种传染性胃肠炎病毒(TGEV)，命名为TGEV 1000(B.Young，K.M.Curtis和R.S.Baric，J.Virol)。74：10600-10611,2000年)。构建了含有绿色荧光蛋白(GFP)基因的重组TGEV，并用其取代了开放阅读框(ORF)3A。将重组TGEV(TGEV-GFP2)转入新生仓鼠肾(BHK)细胞，获得了能高效复制并表达GFP的重组TGEV。构建的复制子构建缺乏ORF 3B和E基因或ORF 3B、E和M基因[分别为TGEV-Rep(AvrII)和TGEV-Rep(EcoNI)]。由于E蛋白和M蛋白是TGEV病毒粒子萌发所必需的，这些复制子RNA应该复制，但不会导致感染性病毒的产生。将携带GFP的复制子RNA与BHK细胞共转染后，荧光显微镜下观察到GFP的表达，逆转录聚合酶链式反应(RT-PCR)检测到携带GFP的先导转录产物。随后将细胞培养上清液传代到允许的猪睾丸(ST)细胞上，没有导致病毒、GFP表达或含有Leader的亚基因组转录本的存在，证明了TGEV复制子RNA的单击性。为制备TGEV复制子颗粒组装包装系统，将TGEV E基因克隆到委内瑞拉马脑炎(VEE)复制子表达载体中，分离出编码TGEV E蛋白的VEE复制子颗粒[VEE-TGEV(E)]。将TGEV-Rep(AvrII)(E基因缺失)与Vee-TGEV(E)RNA转录本共转染BHK细胞，或将TGEV-Rep(AvrII)RNA转录本导入BHK细胞，然后感染携带TGEV E基因的Vee VRPs。在这两种情况下，在转基因细胞中都检测到GFP表达和含有Leader的GFP转录本。细胞培养上清，类似于36小时后收集，被传给新鲜的ST细胞，在那里GFP的表达明显类似于18小时后。RT-PCR检测含有ORF 313和E基因缺失的先导GFP转录本。未从这些培养物中释放重组TGEV。在相同的条件下，TGEV-GFP2在ST细胞培养中扩散，表达GFP，并形成病毒空斑。传染性TGEV复制子颗粒的开发应有助于TGEV复制和组装的研究，并有助于新型猪候选疫苗的生产。
We have recently isolated a transmissible gastroenteritis virus (TGEV) infectious construct designated TGEV 1000 (B. Yount, K. M. Curtis, and R. S. Baric, J. Virol. 74:10600-10611, 2000). Using this construct, a recombinant TGEV was constructed that replaced open reading frame (ORF) 3A with a heterologous gene encoding green fluorescent protein (GFP). Following transfection of baby hamster kidney (BHK) cells, a recombinant TGEV (TGEV-GFP2) was isolated that replicated efficiently and expressed GFP. Replicon constructs were constructed that lacked either the ORF 3B and E genes or the ORF 3B, E, and M genes [TGEV-Rep(AvrII) and TGEV-Rep(EcoNI), respectively]. As the E and M proteins are essential for TGEV virion budding, these replicon RNAs should replicate but not result in the production of infectious virus. Following cotransfection of BHK cells with the replicon RNAs carrying gfp, GFP expression was evident by fluorescent microscopy and leader-containing transcripts carrying gfp were detected by reverse transcription-PCR (RT-PCR). Subsequent passage of cell culture supernatants onto permissive swine testicular (ST) cells did not result in the virus, GFP expression, or the presence of leader-containing subgenomic transcripts, demonstrating the single-hit nature of the, TGEV replicon RNAs. To prepare a packaging system to assemble TGEV replicon particles (TGEV VRP), the TGEV E gene was cloned into a Venezuelan equine encephalitis (VEE) replicon expression vector and VEE replicon particles encoding the TGEV E protein were isolated [VEE-TGEV(E)]. BHK cells were either cotransfected with TGEV-Rep(AvrII) (E gene deletion) and VEE-TGEV(E) RNA transcripts or transfected with TGEV-Rep(AvrII) RNA transcripts and subsequently infected with VEE VRPs carrying the TGEV E gene. In both cases, GFP expression and leader-containing GFP transcripts were detected in transfected cells. Cell culture supernatants, collected similar to36 h posttransfection, were passed onto fresh ST cells where GFP expression was evident similar to18 h postinfection. Leader-containing GFP transcripts containing the ORF 313 and E gene deletions were detected by RT-PCR. Recombinant TGEV was not released from these cultures. Under identical conditions, TGEV-GFP2 spread throughout ST cell cultures, expressed GFP, and formed viral plaques. The development of infectious TGEV replicon particles should assist studies of TGEV replication and assembly as well as facilitate the production of novel swine candidate vaccines.