molecular therapeutic approach for human papillomavirus infections

我们一直在研究以HPV感染为标志物进行宫颈癌分子诊断的可能性。首先，我们证明了在晚期宫颈癌中分离到HPV16型DNA的病例中，可以从患者的血清中分离出针对HPV16E7蛋白的抗体(Fujii等人：JPN癌症杂志86：28-34,1995)。我们还通过刮取已发现宫颈癌前病变的患者的宫颈来收集细胞，结果显示，RT-PCR检测到E6/E7转录产物的比率随着病变的进展而上升(Fujii等：Gyneol Oncol58：210-215,1995)。这些发现表明，E6/E7基因产物在临床上对癌症的发生和维持也有很大的贡献。由于E2蛋白是一种调节E6/E7基因表达的dna结合性蛋白，因此在分析病毒c-…的发病机制时，它是一种非常重要的蛋白。更多的致癌作用。因此，本研究的目的是分离一种能与E2蛋白结合并抑制其转录功能的多肽。在大肠杆菌中表达了HPV16E2蛋白，并对其进行了纯化。利用在噬菌体纤维蛋白中表达随机氨基酸序列的文库，在平板上将E2蛋白转化为固相，获得与E2蛋白结合的噬菌体克隆。结果分离到一个含有色氨酸的多肽序列，当色氨酸被丙氨酸取代时，与E2蛋白的结合比对表明与突变的多肽的结合减少。我们还制备了一种合成肽，将核转位信号添加到分离的多肽序列中，通过荧光素酶活性检测其对E2蛋白转录活性的影响，结果显示转录活性降低。这些发现表明，已分离的多肽与E2蛋白结合并较少地抑制其转录活性

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