TISSUE LOCALIZATION OF SIV EXPRESSING GREEN FLUORESCENT PROTEIN

SIV的一般致病机制研究及早期鉴定 特别是病毒感染的目标细胞将大大 通过容易地识别受感染细胞的技术来促进。冲向 为此，微生物部与 病理分部已经构建了一种重组病毒，能够 体内表达增强型绿色荧光蛋白(EGFP)。绿色荧光蛋白 之所以被选中，是因为该基因很小(717个碱基)，允许准备好 插入SIVmac239的适当缺失突变体。这个 将绿色荧光蛋白插入SIVmac239变异体的nef基因座 在nef基因中包含了一个大的缺失，产生了一种病毒 在CEMx174细胞中快速复制并表达EGFP，导致 细胞荧光强，不添加任何发色原。 当p27gag表达时，90%以上的培养细胞表达绿色荧光蛋白 达到了顶峰。绿色荧光蛋白在该细胞中的表达稳定。 通过重组病毒的多个通道。要检测 体内的免疫表型病毒阳性细胞，我们已经修改了我们的 现有的超低温保存技术以增强GFP和 与抗肿瘤单抗结合的二次染色质 细胞表位。在体内，我们在CD4+中检测到丰富的病毒 外周淋巴结内淋巴细胞与CFA肉芽肿 感染动物的时间范围与未观察到的类似 表达绿色荧光蛋白的病毒。

Studies of SIV pathogenesis in general and identification of early target cells of viral infection in particular would be greatly facilitated by techniques to readily identify infected cells. Toward this end, the Microbiology Division in collaboration with the Pathology Division has constructed a recombinant virus that is capable of expressing enhanced green fluorescent protein (EGFP) in vivo. EGFP was selected because the gene is small (717 bp) allowing for ready insertion into appropriate deletion mutants of SIVmac239. The insertion of EGFP into the nef locus of a variant of SIVmac239 that contained a large deletion in the nef gene produced a virus that replicated rapidly in CEMx174 cells and expressed EGFP resulting in strong fluorescence of the cells without addition of any chromogen. Over 90% of the cultured cells expressed EGFP when p27 gag expression reached its peak. The expression of EGFP remained stable in this cell line through multiple passages of the recombinant virus. To detect the immunophenotype virus-positive cells in vivo, we have modified our existing cryopreservation techniques to enhance colabeling of GFP and a secondary chromagen bound to monoclonal antibodies directed against cellular epitopes. In vivo we have detected abundant virus in CD4+ lymphocytes within peripheral lymph nodes and CFA granulomas in infected animals in a time frame similar to that observed with non EGFP-expressing virus.