HPV16E6、HPV18E7原癌蛋白免疫组化试剂盒的建立

发布时间：2018-01-26 23:35

本文关键词： HPV16E6蛋白 HPV18E7蛋白宫颈癌早期诊断免疫组化　出处：《重庆理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：宫颈癌是由人乳头瘤病毒(Human papllomavirus,HPV)引起的一种常见的高发于女性的恶性肿瘤,其死亡率高居女性肿瘤第二位。近年来统计发现,我国农村地区宫颈癌筛查范围覆盖低,农村地区发病率和致死率均高于城市,并呈逐年增长趋势且发病年龄趋向年轻化,严重威胁女性健康。高危型HPV呈持续感染,尤其是HPV16和HPV18两个型,是导致宫颈癌的根本原因。此外,E6和E7蛋白的过表达在细胞恶性转化中起到至关重要的作用,为潜在的宫颈癌早期诊断标志物。目的:目前我国尚无商业化的基于HPV16E6和HPV18E7蛋白的免疫组化试剂盒,用于检测临床样本中的HPV16和HPV18。用于临床HPV16和HPV18检测的多为HPV DNA检测,不利于资源匮乏地区推广。为此,建立拥有自主知识产权的,基于HPV16E6和HPV18E7蛋白的免疫组化试剂盒更具优势。我们的目的是,以HPV16E6和HPV18E7蛋白为诊断标志物,建立特异性好、成本低的HPV16E6和HPV18E7蛋白免疫组化试剂盒,用于宫颈癌的早期诊断。方法:本课题采用经鉴定的HPV16E6和HPV18E7重组蛋白为抗原,分别用于抗HPV16E6和HPV18E7单克隆抗体的制备。并基于抗HPV16E6和抗HPV18E7单抗,分别建立宫颈癌细胞系的HPV16E6和HPV18E7蛋白检测免疫组化试剂盒。在此基础上,将建立的免疫组化试剂盒应用于临床样本的检测。结果:(1)以纯化的HPV16E6和HPV18E7蛋白为抗原免疫小鼠,经过亚克隆筛选,分别得到了7株和8株可稳定分泌特异性高、亲和性好的抗HPV16E6单抗和抗HPV18E7单抗的杂交瘤细胞株,并分别选择效价最高的属Ig G亚类单抗的HPV16E6-4D4、HPV18E7-E11细胞株进行单抗的制备与鉴定;(2)基于HPV16E6-4D4、HPV18E7-E11单克隆抗体,分别初步建立了宫颈癌细胞系的HPV16E6和HPV18E7蛋白免疫组化检测试剂盒;(3)用所建立的HPV16和HPV18蛋白免疫组化检测试剂盒分别检测临床样本,都成功检测出HPV16E6和HPV18E7蛋白表达阳性,与宫颈癌病理检查结果的符合率分别达95.23%和100%,均显示了良好的诊断性能与潜力。结论:本实验初步建立了HPV16E6和HPV18E7蛋白免疫组化检测试剂盒,并用于不同时期的宫颈癌或癌前病变组织临床样本检测,其具有成本低、特异性好的特点,可用于宫颈癌的早期诊断。
[Abstract]:Cervical cancer is a common malignant tumor in women caused by human papillomavirus (HPV). In recent years, it is found that the scope of cervical cancer screening in rural areas in China is low, the morbidity and mortality in rural areas are higher than those in urban areas. And the disease age tends to be younger, which is a serious threat to the health of women. The high-risk type of HPV is persistent infection, especially HPV16 and HPV18. In addition, the overexpression of E6 and E7 proteins plays a crucial role in the malignant transformation of cells. Objective: there is no commercial immunohistochemical kit based on HPV16E6 and HPV18E7 protein in China. For the detection of HPV16 and HPV18 in clinical samples. Most of the clinical HPV16 and HPV18 detection is HPV DNA detection, which is not conducive to the promotion of resource-poor areas. The establishment of HPV16E6 and HPV18E7 protein based immunohistochemical kits with independent intellectual property rights is more advantageous. Our aim is. Using HPV16E6 and HPV18E7 proteins as diagnostic markers, an immunohistochemical kit of HPV16E6 and HPV18E7 proteins with good specificity and low cost was established. Methods: the recombinant proteins of HPV16E6 and HPV18E7 were used as antigen. It was used in the preparation of monoclonal antibodies against HPV16E6 and HPV18E7, respectively, and was based on anti HPV16E6 and anti HPV18E7 monoclonal antibodies. The HPV16E6 and HPV18E7 protein detection kits of cervical cancer cell lines were established respectively. Results the purified HPV16E6 and HPV18E7 proteins were used as antigen to immunize mice. Seven and eight hybridoma cell lines with stable secretion specificity and good affinity were obtained, respectively. The HPV16E6-4D4D4 HPV18E7-E11 cell line with the highest titer of Ig G subclass McAb was selected to prepare and identify the McAb. (2) based on monoclonal antibody HPV16E6-4D4, HPV18E7-E11. HPV16E6 and HPV18E7 protein immunohistochemical detection kits of cervical cancer cell lines were established. The positive expression of HPV16E6 and HPV18E7 protein was detected successfully by using the established HPV16 and HPV18 protein immunohistochemical detection kit to detect the clinical samples. The coincidence rate with pathological examination of cervical cancer was 95.23% and 100% respectively. Conclusion: the HPV16E6 and HPV18E7 protein immunohistochemical detection kit was established in this experiment. It can be used in the early diagnosis of cervical cancer because of its low cost and good specificity.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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