人乳头瘤病毒18型类病毒颗粒疫苗研制、中和抗体筛选及冷冻电镜三维结构重建

发布时间：2018-03-30 03:30

本文选题：大肠杆菌表达系统　切入点：人乳头瘤病毒18型　出处：《厦门大学》2008年硕士论文

【摘要】： 高危型人乳头瘤病毒(Human Papillomavirus,HPV)病毒感染被认为是引起女性宫颈癌的主要原因。流行病学调查显示98%的宫颈癌都是由人乳头瘤病毒(HPV)感染引起,其中约70%是由HPV16或HPV18引起。因此,开发一种安全,有效的HPV18疫苗对于预防宫颈癌具有重大的意义。首先,本研究构建了高效HPV18L1蛋白大肠杆菌原核表达系统。利用该表达系统,HPV18 L1蛋白能够以可溶性形式表达,保留了其天然的表位。其次,本研究还还针对大肠杆菌来源的HPV18 L1蛋白建立了一套高效,易于放大的纯化工艺,以及类病毒颗粒体外组装工艺。根据该工艺所制备的HPV18VLPs疫苗纯度达到98%以上,各项鉴定指标均符合药典要求。且动物实验显示,该疫苗具有高度的免疫原性,在实验动物体能能诱导高滴度的HPV18中和抗体。本研究在成功制备HPV18 VLPs疫苗的基础上,利用假病毒中和模型,一共鉴定出HPV18中和单抗6株,而后通过阻断实验进一步确定了优势中和单抗11A9以及3C3。为了进一步阐明11A9单抗的中和机制,结合位点,在本研究对HPV18 VLPs进行了冷冻电镜观测,初步了解HPV 18 VLPs的结构模式。此外,还进一步制备了11A9-HPV18 VLPs抗原抗体复合物以备进一步进行冷冻电镜研究。总之,本研究利用大肠杆菌表达系统制备了一种廉价,有效的HPV18 VLP疫苗,对HPV18的中和单抗进行鉴定,并且对于HPV18优势单抗11A9的中和机制,结合位点进行了初步探索,为将来进行HPV18疫苗的分子设计提供了参考。
[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.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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