TISSUE LOCALIZATION OF SIV EXPRESSING GREEN FLUORESCENT PROTEIN

表达绿色荧光蛋白的 SIV 的组织定位

• 批准号: 6591340
• 负责人: VITO G SASSEVILLE
• 金额: $ 11.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591340
• 关键词: AIDS; Mammalia; animal tissue; communicable diseases; hematology; immunology; inflammation; lymphatic system; skin; virus

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 th e recombinant virus To detect the immunophenotype virus-positive cells in vivo, we have modified our existing cryppreservation 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 Publication Alexander, L, Veazey RS, Czajak S, DeMaria M, Rosenzweig M, Lackner AA, Desrosiers RC, Sasseville, VG Recombinant simiam immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys AIDS Res Hum Retroviruses 15:11-21, 1999

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 为此，微生物学司与 病理科构建了一种重组病毒，能够 增强型绿色荧光蛋白（EGFP）的体内表达 选择它是因为它的基因很小（717 bp）， 插入到SIVmac239的适当缺失突变体中 将EGFP插入SIVmac239变体的nef基因座中， nef基因的大缺失产生了一种病毒， 在CEMx174细胞中快速表达EGFP， 不添加任何色原的细胞荧光超过90% 当p27 gag表达达到 EGFP在该细胞系中的表达保持稳定 通过重组病毒的多次传代， 免疫表型病毒阳性细胞在体内，我们已经修改了我们的 现有的密码保存技术，以增强GFP的共同标记， 与单克隆抗体结合的二级发色团， 细胞表位在体内，我们已经检测到丰富的病毒在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表达病毒出版物亚历山大，L，Veazey RS，Czajak S， DeMaria M，Rosenzweig M，Lackner AA，Desrosiers RC，Sasseville，VG 表达绿色荧光的重组猴免疫缺陷病毒 蛋白质识别恒河猴中受感染的细胞 逆转录病毒15：11 - 21，1999