人乳头瘤病毒（HPV）衣壳蛋白L1的体外可控组装

发布时间：2018-12-11 00:58

【摘要】：人乳头瘤病毒(HPV)是一类能够感染皮肤和粘膜的DNA病毒。高危型HPV是宫颈癌以及癌前病变的主要致病因子。目前，，在临床上没有治疗宫颈癌的恰当措施，因此预防性HPV疫苗的研制尤为重要。虽然已经有两种预防性病毒样颗粒（VLPs）疫苗被批准上市，但是它们不仅稳定性差、造价高，而且具有免疫上严格的型别特异性，所以廉价、高效的第二代广谱疫苗的研发势在必行。本论文以降低HPV疫苗成本和提高其广谱性为出发点，以揭示HPV衣壳蛋白L1自组装机理和关键组装因素为目的，主要开展了由单体到五聚体、再到VLPs的体外可控组装过程研究。主要内容如下：（一）、在大肠杆菌表达体系中，我们以L1五聚体形成VLPs的分子机制为基础，设计、构建并表达了两个具有新型结构的重组L1蛋白——HPV16L1Bi和HPV18L1Bi。在原有的分子间作用力的驱动下首次成功地制备出一种新型的、同时含有两种HPV型别的杂合HPV16/18L1Bi VLPs。利用蔗糖密度梯度离心和免疫共沉淀(Co-IP)的方法进一步纯化得到了VLPs的纯杂合子，其中HPV16和HPV18L1五聚体的比例约是3:5。杂合的VLPs针对HPV16和HPV18型别均能诱导产生高滴度的中和抗体，具有这两个型别的免疫原性。本研究为含有多种型别杂合VLPs疫苗的研发提供了新思路，也为生物大分子自组装过程的调控和关键因素的揭示奠定了理论基础。（二）、在上述研究中，我们还发现所有型别乳头瘤病毒L1蛋白C-末端α-螺旋5（h5）序列中的LGR具有绝对保守性。利用基因工程技术把HPV16L1蛋白序列中的464LGR466分别被点突变成丙氨酸（Ala, A）后所表达的L1蛋白均仅以单体形式存在，无法再组装成五聚体。表明LGR是形成L1五聚体必不可少的氨基酸，这是迄今为止所发现的阻断L1五聚体形成的最小结构单元。基于此，我们以HPV16L1蛋白h5为靶标设计合成了一条包括h5在内的15肽，它能够有效地抑制L1五聚体的形成，表明h5是潜在的抗病毒靶点，可以以此制备出以h5为结构基础的靶向性小肽药物。另外，HPV16L1蛋白469位的亮氨酸（Leu, L）被突变成丙氨酸后，其可溶性五聚体的产量至少增加1倍。本研究为抗HPV药物的研发以及降低HPV疫苗成本奠定了基础。
[Abstract]:Human papillomavirus (HPV) is a class of DNA viruses that can infect skin and mucosa. High risk HPV is the main pathogenic factor of cervical cancer and precancerous lesions. At present, there is no clinical treatment for cervical cancer, so the development of prophylactic HPV vaccine is particularly important. Although two prophylactic virus-like (VLPs) vaccines have been approved for sale, they are not only unstable and expensive, but also have strict type specificity in immunization, so they are cheap. The development of high-efficient second-generation wide-spectrum vaccine is imperative. In order to reduce the cost of HPV vaccine and improve its broad spectrum, and to reveal the mechanism of HPV capsid protein L1 self-assembly and key assembly factors, this paper mainly studied the controllable assembly process from monomer to pentamer to VLPs in vitro. The main contents are as follows: (1) in Escherichia coli expression system, we designed, constructed and expressed two novel recombinant L1 proteins, HPV16L1Bi and HPV18L1Bi., based on the molecular mechanism of L1 pentamer forming VLPs. A novel hybrid HPV16/18L1Bi VLPs. with two HPV types was successfully prepared under the driving of the original intermolecular force for the first time. The homozygote of VLPs was further purified by sucrose density gradient centrifugation and immunoprecipitation (Co-IP), in which the ratio of HPV16 to HPV18L1 was about 3: 5. Heterozygous VLPs can induce the production of high titer neutralizing antibodies against both HPV16 and HPV18 types and has the immunogenicity of these two types. This study provides a new idea for the research and development of VLPs vaccine containing multiple types of heterozygotes. It also lays a theoretical foundation for the regulation of self-assembly process of biological macromolecules and the disclosure of key factors. (2) in the above studies, we also found that LGR in the C-terminal 伪 -helix 5 (H5) sequence of all types of papillomavirus L1 protein is absolutely conserved. The L1 protein expressed after the 464LGR466 of HPV16L1 protein was mutated into alanine (Ala, A) by genetic engineering was expressed in the form of monomers and could not be assembled into pentamers. It is shown that LGR is the essential amino acid to form L1 pentamers, which is the smallest structural unit found so far to block the formation of L1 pentamers. Based on this, we designed and synthesized a 15-peptide including H5, which can effectively inhibit the formation of L1 pentamers, indicating that H5 is a potential antiviral target. Thus, the targeted small peptide drugs based on the structure of H 5 can be prepared. In addition, when leucine (Leu, L) at position 469 of HPV16L1 protein was mutated to alanine, the yield of soluble pentamer increased at least double. This study has laid a foundation for the development of anti-HPV drugs and reducing the cost of HPV vaccine.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33;R392

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