PRODUCTION OF ATTENUATED VACCINE CANDIDATE DENGUE VIRUSES FROM INFECTIOUS CDNA

登革病毒基因组是一种正链RNA分子， 大约11KB。纯化的基因组RNA在转染后具有感染性 放入合适的细胞中。这个项目的目标是克隆一个cDNA拷贝 登革病毒2型的基因组， 基因组RNA的拷贝可以通过体外转录制备。创作 感染性克隆将允许将定义的突变引入到 登革热基因组。 这种能力在许多类型的通信系统中是有用的。 研究，包括绘制导致宿主范围改变的突变 在嗜神经性DEN2菌株中，并试图减弱DEN2， 可以用作疫苗。我们一直采用的策略是 然后使用逆转录/聚合酶链反应 来制造大量的cDNA。我们试图放大相对较大的 基因组的片段，所以只有少数克隆片段必须被 组装成一个完整的副本。我们可以常规扩增3 - 4kb 片段，但我们不能可靠地放大较大的片段。 因此 整个基因组被成功扩增并克隆为四个片段。 其中两个片段很容易结合起来，形成正确的半基因组。 到目前为止，获得左半基因组的努力被证明是不成功的; 迄今为止分离的所有这些克隆都被重排。 我们目前正在 寻求几种替代战略来解决我们的问题：我们是 试图制造正确的四分之三基因组， 通过两段体外连接而无需克隆全长cDNA 整个基因组;我们正试图克隆左半基因组在一个低， E. coli中，然后在 同样的载体，我们用E做实验设计的大肠杆菌菌株， 繁殖“困难”的DNA片段;我们开始尝试克隆， 酵母希望这些方法中的一个或多个很快就会成功。

The dengue virus genome is a positive-strand RNA molecule of approximately 11 kb. Purified genomic RNA is infectious upon transfection into appropriate cells. The goal of this project is to clone a cDNA copy of the genome of dengue virus type 2 such that a faithful, infectious copy of the genomic RNA can be made by in vitro transcription. Creation of an infectious clone will allow introduction of defined mutations into the dengue genome. Such a capability would be useful in many types of studies, including mapping mutations responsible for altered host range in neurotropic strains of DEN2, and attempting to attenuate DEN2 for possible use as a vaccine. The strategy we have been using is to prepare genomic RNA and then use reverse transcription/polymerase chain reaction to make large amounts of cDNA. We have tried to amplify relatively large segments of the genome, so that only a few cloned pieces have to be assembled to make a full-length copy. We can routinely amplify 3-4 kb segments, but we can not reliably amplify larger pieces. Thus, the entire genome has been successfully amplified and cloned as four pieces. Two of the pieces were easily combined to make the right half-genome. Efforts to obtain the left half-genome have so far proved unsuccessful; all such clones isolated to date have been rearranged. We are currently pursuing several alternative strategies to solve our problems: we are attempting to make the right three-quarter-genome, in order to produce a full-length cDNA by two-piece in vitro ligation without having to clone the entire genome; we are trying to clone the left half-genome in a low copy-number vector in E. coli, and then assemble a full-length clone in the same vector; we are experimenting with E. coli strains designed to propagate "difficult" pieces of DNA; we are beginning to try cloning in yeast. Hopefully, one or more of these approaches will soon succeed.