人乳头瘤病毒假病毒中和模型的建立和应用

发布时间：2018-01-20 15:44

本文关键词： 人乳头瘤病毒假病毒中和抗体　出处：《厦门大学》2006年硕士论文　论文类型：学位论文

【摘要】： 人乳头瘤病毒(human papillomavirus, HPV)感染能引起宫颈癌、生殖器疣等多种性传播疾病,严重危害着人类健康。由于HPV的生活周期依赖于上皮细胞的分化,传统的细胞培养方法不能实现HPV的扩增,同时从病人组织中分离的病毒也极其有限,这制约了HPV的分子生物学研究和HPV疫苗的保护性评价。研究人员已经发展出多种系统以克服病毒来源不足的限制:通过组织移植和筏式组织培养扩增一些HPV型的病毒,以及用重组痘病毒、腺病毒、酵母表达构建HPV假病毒。但是这些病毒/假病毒生产方法的操作较复杂,效率均不高,无法满足大量试验的需要。本论文利用HPV研究方面的新进展,结合本研究中心的实际条件,用磷酸钙转染方法将含HPV优化基因的质粒和报告质粒共转染,成功构建获得了高滴度的感染性HPV16、HPV18、HPV6和HPV11四种假病毒,电镜观察证实获得的假病毒具有与天然病毒相同的形态,随后用标准中和单抗进行的鉴定证实假病毒具有与天然病毒相似的感染性。这些结果说明获得的假病毒可以替代天然病毒用于HPV生物学研究和HPV候选疫苗的保护性评价。我们用获得的四种假病毒对29种哺乳动物细胞进行了感染比较。发现HPV16和HPV18假病毒有很广的细胞嗜性,能感染人、啮齿动物和猴不同组织的多种细胞系; HPV6和HPV11假病毒能感染人的多种细胞系,但是基本不感染啮齿动物和猴的细胞。该结果暗示“高危”型HPV和“低危”型HPV对细胞的感染机制可能不同。基于多细胞感染结果,我们选择293FT细胞作为假病毒感染靶细胞,建立了假病毒中和模型。本实验室已经筛选获得了HPV16、HPV18和HPV6的单抗各几十株,我们用假病毒感染中和试验从中鉴定出17株HPV16中和单抗、9株HPV18中和单抗和22株HPV6中和单抗。滴度最高的16株HPV6中和单抗有12株能阻断HPV11假病毒的感染,其中5株单抗对HPV11假病毒的中和滴度能达到104以上,表明HPV6和HPV11具有共同的中和表位。鉴定出的这些中和单抗对于全面鉴定HPV中和表位和HPV疫苗质控均有较大的帮助。
[Abstract]:Human papillomavirus (HPV) infection can cause cervical cancer, genital warts and other sexually transmitted diseases. Because the life cycle of HPV depends on the differentiation of epithelial cells, the traditional methods of cell culture can not achieve the amplification of HPV, and the virus isolated from patients' tissues is very limited. This has limited the molecular biology of HPV and the protective evaluation of HPV vaccines. Researchers have developed multiple systems to overcome the limitations of inadequate sources of the virus:. Some HPV viruses were amplified by tissue transplantation and raft tissue culture. Recombinant poxvirus, adenovirus and yeast were used to construct HPV pseudovirus. However, the production methods of these viruses / pseudoviruses were complex and inefficient, and could not meet the needs of a large number of tests. In this thesis, the plasmid containing the optimized gene of HPV and the reporter plasmid were co-transfected by calcium phosphate transfection method, based on the new development of HPV research and the actual conditions of this research center. Four pseudoviruses, HPV18 HPV6 and HPV11, were successfully constructed with high titer of HPV16 and HPV18HPV6. Electron microscopy showed that the pseudoviruses had the same morphology as natural viruses. The identification of pseudoviruses with standard neutralization and McAbs proved that pseudoviruses are similar to natural viruses. These results indicate that the obtained pseudoviruses can replace natural viruses in HPV biological research and HPV candidate disease. Protective evaluation of seedlings. We compared the infection of 29 mammalian cells with four pseudoviruses. We found that HPV16 and HPV18 pseudoviruses have a wide cytotropism and can infect human. A variety of cell lines from different tissues of rodents and monkeys; HPV6 and HPV11 pseudovirus can infect a variety of human cell lines. The results suggest that the mechanism of "high-risk" HPV and "low-risk" HPV infection may be different, based on the results of multicellular infection. We selected 293FT cells as target cells for pseudovirus infection and established a pseudoviral neutralization model. We have screened and obtained dozens of McAb strains of HPV16 HPV18 and HPV6 respectively. We have identified 17 HPV16 neutralizing McAbs from these McAbs by pseudovirus neutralization test. Nine HPV18 neutralizing McAbs and 22 HPV6 neutralizing McAbs. 12 of 16 HPV6 neutralizing McAbs with the highest titers could block the infection of HPV11 pseudovirus. The neutralization titer of 5 McAbs to HPV11 pseudovirus was over 104. The results showed that HPV6 and HPV11 had the same neutralization epitopes, and the identified neutralization McAbs were helpful for the identification of HPV neutralizing epitopes and the quality control of HPV vaccines.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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