CONSTRUCTION OF INFECTIOUS FULL LENGTH CDNA CLONE OF DENGUE VIRUS TYPE 1

去年我们报道了我们成功地克隆了一个全长的cdna。 登革2型病毒(DEN2)，其传染性转录物可能来自于 制造。今年，我们已经开始了一项合作，与 另一种登革热血清型，DEN1。描述了到目前为止完成的工作 下面。扩增出DEN1的左侧2.6kb和右侧0.5kb 以DEN1病毒粒子RNA为模板，以耐热DNA为模板进行RT-PCR 聚合酶，错误率低。用于聚合酶链式反应的引物 在DEN1 5‘端上游加入了SP6启动子和一个 独特的SAC II场地就在3‘端的下游。这两个cDNAs 将片段克隆到酵母穿梭载体的多联体中 PRS424，定向正确。这种DNA是用一种酶线性化的 切断了介于两个dDNA之间的多聚链接物。与此同时，一个8kb的 通过RT-PCR从DEN1 RNA中获得了cDNA，使用了一种更容易出错的 能够进行长时间聚合酶扩增的聚合酶。这个“填充片段”表示 位于DEN1基因组的中间，并与左侧和右侧重叠 包含在PRS克隆中的cDNA。填充片段和 线性化的PrS DNA用于将感受态Trp-酵母共转化到Trp+。 这选择了携带TRP1的PRS质粒的环状形式 赋予Trp+表型所必需的基因。在酵母中，双同系物 填充物片段与左手和 右手边的cDNA创建了一个包含 全长DEN1基因。用聚合酶链式反应筛选Trp+酵母菌落 确认它们携带的PrS质粒的结构。质粒 将明显含有全长DEN1基因的基因导入E. Coli，并进行大规模DNA制备。其中四个的结构 经限制性内切酶鉴定，证实这些大肠杆菌来源的质粒正确。 分析。从这四个克隆得到的转录本被电穿孔 进入哺乳动物细胞，但不具传染性。随后的分析由 利用痘苗病毒-T7RNA聚合酶系统进行瞬时表达 表明这四个克隆没有形成预期的DEN1模式 蛋白质。对其中两个克隆的选定区域进行了测序 完成，并且显示填充片段序列中存在错误， 在一种情况下包括一个终止密码子。因此，我们的长聚合酶链式反应产物可能不会 做一份忠实的复制品。我们将继续筛选更多的全长 寻找可能产生传染性转录本的克隆。同时， 我们计划对一个或多个非传染性克隆进行完全测序，并 然后尝试用错误率低的cDNA来修复错误 以聚合酶作为修复基因的来源。

Last year we reported our success in making a full-length cDNA clone of dengue virus type 2 (DEN2) from which infectious transcripts could be made. This year we have begun a collaboration to repeat this with another dengue serotype, DEN1. The work accomplished so far is described below. The left-hand 2.6 kb and right-hand 0.5 kb of DEN1 were amplified by RT-PCR using DEN1 virion RNA as template and a thermostable DNA polymerase with a low error rate. The primers used for the PCR incorporated an SP6 promoter just upstream of the DEN1 5' end and a unique Sac II site just downstream of the 3' end. These two cDNA fragments were cloned into the polylinker of the yeast shuttle vector pRS424, oriented properly. This DNA was linearized with an enzyme that cuts in the polylinker in between the two DEN cDNAs. Meanwhile, an 8 kb cDNA was made from DEN1 RNA by RT-PCR, using a more error-prone polymerase capable of long PCR. This "stuffer fragment" represents the middle of the DEN1 genome and overlaps both the left- and right-hand cDNAs contained in the pRS clone. The stuffer fragment and the linearized pRS DNA were used to cotransfect competent trp- yeast to TRP+. This selects for circular forms of the pRS plasmid, which carries theTRP1 gene necessary to confer the TRP+ phenotype. In yeast, double homologous recombination between the stuffer fragment and the left-hand and right-hand cDNAs creates a circular pRS plasmid containing the full-length DEN1 cDNA. TRP+ yeast colonies were screened by PCR to confirm the structure of the pRS plasmids they harbored. Plasmids apparently containing full-length DEN1 cDNA were transfected into E. coli, and large scale DNA preps were done. The structures of four of these E. coli derived plasmids were confirmed as correct by restriction analysis. Transcripts made from these four clones were electroporated into mammalian cells, but were not infectious. Subsequent analysis by transient expression using the vaccinia virus - T7 RNA polymerase system showed that these four clones did not make the expected pattern of DEN1 proteins. Sequencing of selected regions of two of these clones has been done, and shows that there are errors in the stuffer fragment sequences, including a stop codon in one case. Thus, our long PCR product may not be a faithful copy. We will continue to screen for more full-length clones to seek ones that might make infectious transcripts. Meanwhile, we plan to fully sequence one or more of the noninfectious clones, and then try to repair the mistakes, using cDNA made with the low error rate polymerase as the source of the cDNA for repair.