本文選題：大腸桿菌表達系統(tǒng)　切入點：人乳頭瘤病毒18型　出處：《廈門大學(xué)》2008年碩士論文

【摘要】： 高危型人乳頭瘤病毒(Human Papillomavirus,HPV)病毒感染被認為是引起女性宮頸癌的主要原因。流行病學(xué)調(diào)查顯示98%的宮頸癌都是由人乳頭瘤病毒(HPV)感染引起,其中約70%是由HPV16或HPV18引起。因此,開發(fā)一種安全,有效的HPV18疫苗對于預(yù)防宮頸癌具有重大的意義。 首先,本研究構(gòu)建了高效HPV18L1蛋白大腸桿菌原核表達系統(tǒng)。利用該表達系統(tǒng),HPV18 L1蛋白能夠以可溶性形式表達,保留了其天然的表位。 其次,本研究還還針對大腸桿菌來源的HPV18 L1蛋白建立了一套高效,易于放大的純化工藝,以及類病毒顆粒體外組裝工藝。根據(jù)該工藝所制備的HPV18VLPs疫苗純度達到98%以上,各項鑒定指標(biāo)均符合藥典要求。且動物實驗顯示,該疫苗具有高度的免疫原性,在實驗動物體能能誘導(dǎo)高滴度的HPV18中和抗體。 本研究在成功制備HPV18 VLPs疫苗的基礎(chǔ)上,利用假病毒中和模型,一共鑒定出HPV18中和單抗6株,而后通過阻斷實驗進一步確定了優(yōu)勢中和單抗11A9以及3C3。為了進一步闡明11A9單抗的中和機制,結(jié)合位點,在本研究對HPV18 VLPs進行了冷凍電鏡觀測,初步了解HPV 18 VLPs的結(jié)構(gòu)模式。此外,還進一步制備了11A9-HPV18 VLPs抗原抗體復(fù)合物以備進一步進行冷凍電鏡研究。 總之,本研究利用大腸桿菌表達系統(tǒng)制備了一種廉價,有效的HPV18 VLP疫苗,對HPV18的中和單抗進行鑒定,并且對于HPV18優(yōu)勢單抗11A9的中和機制,結(jié)合位點進行了初步探索,為將來進行HPV18疫苗的分子設(shè)計提供了參考。
[Abstract]:High risk human papillomavirus (HPV) infection is considered to be the leading cause of cervical cancer in women. Epidemiological studies show that 98% of cervical cancers are caused by HPV infection, of which about 70% are caused by HPV16 or HPV18. The development of a safe and effective HPV18 vaccine is of great significance for the prevention of cervical cancer. First of all, a prokaryotic expression system of HPV18L1 protein was constructed, by which the HPV18 L1 protein could be expressed in a soluble form and its natural epitope was preserved. Secondly, this study also established a set of high efficiency and easy amplification purification technology for HPV18 L1 protein from Escherichia coli, as well as an in vitro assembly process of viroid particles. The purity of HPV18VLPs vaccine prepared by this process was over 98%. All the identification indexes met the requirements of pharmacopoeia and the animal experiments showed that the vaccine had high immunogenicity and could induce high titer of HPV18 neutralizing antibodies in laboratory animals. Based on the successful preparation of HPV18 VLPs vaccine, six strains of HPV18 neutralizing monoclonal antibody were identified by using pseudovirus neutralization model. In order to further elucidate the neutralization mechanism of 11A9 McAbs, the HPV18 VLPs was observed by freezing electron microscope in this study, in order to further elucidate the neutralization mechanism of 11A9 McAbs, and to further clarify the neutralization mechanism of 11A9 McAbs. The structural pattern of HPV 18 VLPs was preliminarily understood. In addition, 11A9-HPV18 VLPs antigen-antibody complexes were prepared for further study by cryopreservation electron microscopy. In conclusion, a cheap and effective HPV18 VLP vaccine was prepared by using E. coli expression system to identify the neutralizing monoclonal antibody of HPV18, and the neutralization mechanism of HPV18 dominant monoclonal antibody 11A9 was preliminarily explored. It provides a reference for the molecular design of HPV18 vaccine in the future.
【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】：2008
【分類號】：R392

【引證文獻】

相關(guān)博士學(xué)位論文 前1條

1 孫博;應(yīng)用昆蟲細胞表達預(yù)防性人乳頭瘤病毒疫苗和應(yīng)用填充床生物反應(yīng)器表達H1N1流感病毒疫苗的研究[D];吉林大學(xué);2012年

，

本文編號：1684146