MONITORING FOREIGN GENE-EXPRESSION BY A HUMAN ADENOVIRUS-BASED VECTOR USING THE FIREFLY LUCIFERASE GENE AS A REPORTER
MONITORING FOREIGN GENE-EXPRESSION BY A HUMAN ADENOVIRUS-BASED VECTOR USING THE FIREFLY LUCIFERASE GENE AS A REPORTER
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DOI：
10.1016/0168-1702(93)90090-a
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发表时间：
1993-04-01
期刊：
影响因子：
5
通讯作者：
GRAHAM, FL
中科院分区：
文献类型：
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作者：
MITTAL, SK;MCDERMOTT, MR;GRAHAM, FL
我们构建了一个辅助独立的腺病毒型5-荧光素酶重组体（Ad5- luc 3），该重组体含有萤火虫荧光素酶基因，其两侧是插入到Ad5基因组早期3区（E3）的猿猴病毒40 （SV40）调控序列。在Ad5-Luc3感染的细胞中，荧光素酶的表达相对高效。在HeLa细胞中，感染后36小时，每10(6)个细胞产生大约20马克荧光素酶，在[S-35]甲硫氨酸标记的Ad5-Luc -3感染细胞提取物中，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）直接分析，可以清晰地看到一条62千道尔顿（kDa）的荧光素酶带。在存在或不存在1- β - d -阿拉伯糖醛基胞嘧啶（AraC）的情况下，用Ad5-Luc - 3感染培养细胞的实验结果表明，大多数荧光素酶的表达依赖于病毒DNA复制。这表明该酶可能主要是从Ad5主要晚期启动子表达的转录本衍生的mRNA翻译而来的。在家兔中培养抗荧光素酶抗体，并通过免疫沉淀和Western blot分析进一步表征Ad5-Luc 3感染HeLa细胞中表达的荧光素酶。通过脉冲追踪分析，计算出Ad5-Luc 3感染HeLa细胞中表达的荧光素酶的半衰期约为6-8 h。荧光素酶可能是监测实验动物病毒传播和基因表达的有用标记物，因为酶活性测定方法非常敏感，而且在所有组织中背景值都很低。腹腔注射Ad5-Luc - 3的小鼠，肝脏、脾脏、肾脏和肺部检测荧光素酶活性。单次注射Ad5- luc - 3小鼠足以提高抗荧光素酶抗体和Ad5中和抗体，并持续至少8周。即使存在循环抗荧光素酶和Ad5中和抗体，在第二次口服Ad5- luc 3的小鼠的肝脏、脾脏和肾脏中也可以检测到荧光素酶的活性。
We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 mug luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [S-35]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.