Holterman L, Dubbes R, Mullins J, Haaijman J, Heeney J
Department of Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands.
J Virol Methods. 2000 Jan;84(1):37-48. doi: 10.1016/s0166-0934(99)00136-6.
To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.
为了对艾滋病感染和发展过程中出现的慢病毒变体进行生物学特性分析，人们开发了一种方法，可从感染恒河猴的血清中低至20微升的循环猴免疫缺陷病毒（SIV）颗粒构建分子克隆。该技术使用一种长距离RT-PCR方法，该方法经过优化，可用于扩增部分重叠的5-kb SIV（半基因组）扩增子。基因组两半的连接导致全长克隆的构建，转染后，这些克隆能够在恒河猴外周血单核细胞（PBMC）和各种诱导合胞体的人T细胞系中良好复制。除了研究因免疫逃逸而在循环中出现的分子克隆病毒准种外，该方法还可用于获得完整基因或全长分子克隆。这些克隆可能存在于其他细胞外体液中，如尿液、唾液、眼泪、淋巴液以及支气管或脑脊液。以这种方式扩增的基因可以快速插入新的重组表达载体中，然后可应用于DNA疫苗接种方法。
