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molecular therapeutic approach for human papillomavirus infections

人乳头瘤病毒感染的分子治疗方法

基本信息

批准号：
13671746
负责人：
FUJII Takuma
金额：
$ 2.3万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671746/
关键词：
transcriptional factor virus phage peptides genome transcriptional activity anti-viral agents cervical cancer

项目摘要

We have been investigating the possibility of molecular diagnosis of cancer of the uterine cervix by using HPV infection as a marker. First, we demonstrated that there are cases of advanced cancer of the cervix in which HPV16 type DNA is isolated in which it is possible to isolate antibody to HPV16 E7 protein from the patient's serum (Fujii et al.: Jpn J Cancer Res 86:28-34, 1995). We also collected cells by scraping the uterine cervix of patients in whom precancerous lesions of cervical cancer had been found and showed that the rate of detection of E6/E7 transcription products by RT-PCR rose as the lesions became more advanced (Fujii et al.: Gynecol Oncol 58:210-215, 1995). These findings showed that E6/E7 gene products greatly contribute to the development and maintenance of cancer clinically as well. Since E2 protein is a DNA-binding protein that modulates expression of the E6/E7 genes, it can be concluded to be a very important protein in terms of analyzing the mechanism of viral c … More arcinogenesis. The purpose of the present study was therefore to isolate a peptide that binds to E2 protein and inhibits its transcription function. HPV16 E2 protein was expressed in E. coli, and the protein was purified. The E2 protein was converted to the solid phase on plates, and a phage clone that binds to E2 protein was isolated by using a library in which random amino acid sequences were expressed in phage fiber proteins. A peptide sequence containing tryptophan was isolated as a result, and when the tryptophan was replaced with alanine, comparison of binding to E2 protein showed that binding was decreased with the mutant peptide. We also prepared a synthetic peptide in which the nuclear translocation signal was added to the peptide sequence that was isolated, and assessment of its effect on E2 protein transcription activity by the luciferase assay showed a reduction in transcription activity. These findings demonstrated that the peptide that had been isolated binds to E2 protein and suppresses its transcription activity Less

我们一直在研究利用HPV感染作为标记物对子宫颈癌进行分子诊断的可能性。首先，我们证明，在分离出HPV16型DNA的晚期宫颈癌病例中，有可能从患者血清中分离出HPV16 E7蛋白抗体（Fujii等人：Jpn J cancer Res 86:28- 34,1995）。我们还通过刮拭宫颈癌癌前病变患者的宫颈收集细胞，发现随着病变的进展，RT-PCR对E6/E7转录产物的检出率上升（Fujii et al.: Gynecol Oncol 58:210- 2155,1995）。这些研究结果表明，E6/E7基因产物在临床上也对癌症的发生和维持起着重要作用。E2蛋白是一种调节E6/E7基因表达的dna结合蛋白，在分析病毒c…More癌变机制方面具有重要意义。因此，本研究的目的是分离一种与E2蛋白结合并抑制其转录功能的肽。在大肠杆菌中表达了hpv16e2蛋白，并纯化了该蛋白。将E2蛋白在平板上转化为固相，利用噬菌体纤维蛋白中表达的随机氨基酸序列文库分离出与E2蛋白结合的噬菌体克隆体。结果分离出含有色氨酸的肽序列，当色氨酸被丙氨酸取代时，与E2蛋白的结合比较表明，与突变肽的结合减少。我们还制备了一个合成肽，将核易位信号添加到分离的肽序列中，并通过荧光素酶试验评估其对E2蛋白转录活性的影响，结果显示转录活性降低。这些结果表明，分离的肽与E2蛋白结合，抑制其转录活性较低