TISSUE LOCALIZATION OF SIV EXPRESSING GREEN FLUORESCENT PROTEIN

SIV致病机理的一般研究和早期SIV的鉴定 特别是病毒感染的靶细胞， 通过技术来容易地识别受感染的细胞。 朝向 为此，微生物学司与 病理学部已经构建了一种重组病毒 在体内表达增强型绿色荧光蛋白（EGFP）。 EGFP 选择它是因为它的基因很小（717 bp）， 插入SIVmac239的适当缺失突变体中。 的 将EGFP插入SIVmac239变体的nef基因座， 在nef基因中有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， 细胞的强荧光而不添加任何色原。 当p27 gag表达时，90%以上的培养细胞表达EGFP 达到了顶峰。 EGFP在该细胞中稳定表达 通过重组病毒的多次传代， 检测 免疫表型病毒阳性细胞在体内，我们已经修改了我们的 现有的低温保存技术可增强GFP的共标记， 与单克隆抗体结合的二级发色团， 细胞表位 在体内，我们已经检测到大量的病毒在CD4+ 外周淋巴结内的淋巴细胞和CFA肉芽肿 感染动物的时间范围与非感染动物的时间范围相似 EGFP表达病毒。

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.