本文關(guān)鍵詞： 人乳頭瘤病毒 假病毒 中和抗體　出處：《廈門大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 人乳頭瘤病毒(human papillomavirus, HPV)感染能引起宮頸癌、生殖器疣等多種性傳播疾病,嚴(yán)重危害著人類健康。由于HPV的生活周期依賴于上皮細(xì)胞的分化,傳統(tǒng)的細(xì)胞培養(yǎng)方法不能實現(xiàn)HPV的擴增,同時從病人組織中分離的病毒也極其有限,這制約了HPV的分子生物學(xué)研究和HPV疫苗的保護(hù)性評價。研究人員已經(jīng)發(fā)展出多種系統(tǒng)以克服病毒來源不足的限制:通過組織移植和筏式組織培養(yǎng)擴增一些HPV型的病毒,以及用重組痘病毒、腺病毒、酵母表達(dá)構(gòu)建HPV假病毒。但是這些病毒/假病毒生產(chǎn)方法的操作較復(fù)雜,效率均不高,無法滿足大量試驗的需要。 本論文利用HPV研究方面的新進(jìn)展,結(jié)合本研究中心的實際條件,用磷酸鈣轉(zhuǎn)染方法將含HPV優(yōu)化基因的質(zhì)粒和報告質(zhì)粒共轉(zhuǎn)染,成功構(gòu)建獲得了高滴度的感染性HPV16、HPV18、HPV6和HPV11四種假病毒,電鏡觀察證實獲得的假病毒具有與天然病毒相同的形態(tài),隨后用標(biāo)準(zhǔn)中和單抗進(jìn)行的鑒定證實假病毒具有與天然病毒相似的感染性。這些結(jié)果說明獲得的假病毒可以替代天然病毒用于HPV生物學(xué)研究和HPV候選疫苗的保護(hù)性評價。 我們用獲得的四種假病毒對29種哺乳動物細(xì)胞進(jìn)行了感染比較。發(fā)現(xiàn)HPV16和HPV18假病毒有很廣的細(xì)胞嗜性,能感染人、嚙齒動物和猴不同組織的多種細(xì)胞系; HPV6和HPV11假病毒能感染人的多種細(xì)胞系,但是基本不感染嚙齒動物和猴的細(xì)胞。該結(jié)果暗示“高�！毙虷PV和“低�！毙虷PV對細(xì)胞的感染機制可能不同。基于多細(xì)胞感染結(jié)果,我們選擇293FT細(xì)胞作為假病毒感染靶細(xì)胞,建立了假病毒中和模型。 本實驗室已經(jīng)篩選獲得了HPV16、HPV18和HPV6的單抗各幾十株,我們用假病毒感染中和試驗從中鑒定出17株HPV16中和單抗、9株HPV18中和單抗和22株HPV6中和單抗。滴度最高的16株HPV6中和單抗有12株能阻斷HPV11假病毒的感染,其中5株單抗對HPV11假病毒的中和滴度能達(dá)到104以上,表明HPV6和HPV11具有共同的中和表位。鑒定出的這些中和單抗對于全面鑒定HPV中和表位和HPV疫苗質(zhì)控均有較大的幫助。
[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.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R373

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本文編號：1448757