人乳头瘤病毒11型L1蛋白的突变分析及其类病毒颗粒疫苗的研制

发布时间：2018-06-20 12:11

本文选题：人乳头瘤病毒11型 + L1蛋白　；参考：《厦门大学》2007年硕士论文

【摘要】： 流行病学和相关研究证实人乳头瘤病毒(HPV)感染与尖锐湿疣、宫颈上皮瘤样增生和宫颈癌的发生关系密切。其中,HPV6、11型是尖锐湿疣的主要致病因子,大约90%的生殖器疣与HPV6和HPV11有关。预防HPV11感染的疫苗研制具有良好的社会效益及经济效益。研究结果表明,HPV L1能独立组装成为HPV类病毒颗粒(VLP),从而良好地再现HPV的天然中和表位,并具有较好的免疫原性,是目前预防性疫苗研究的主要目标蛋白。本研究利用原核表达系统非融合表达HPV11 L1蛋白及其突变体,研究N端序列以及关键氨基酸对HPV11 L1的表达和VLP组装效率的影响,从而获得可用于预防HPV11病毒感染的疫苗。首先,HPV11-L1蛋白N端缺失突变结果表明,N端缺失不超过16aa的突变不影响VLP的形成,但对目的蛋白的表达量有不同程度的影响,其中缺失4aa的突变体HPV11N4C-L1在大肠杆菌中的表达量较大,可用于预防性疫苗的研究。其次,分析Genebank中两株HPV11序列发现在aa364、aa453、aa501出现差异(分别为Glu~(364)和Lys~(364)、Ser~(453)和Tyr~(453)、Lys~(501)和Arg~(501)),实验显示两种蛋白的理化性质和VLP的组装有较大的差异。定点突变实验表明,aa453与aa501的差异并不影响蛋白的性质,但aa364对VLP的组装效率至关重要。进一步将aa364突变成为Gln、Asp、Arg、Phe、Gly和Leu的实验表明,氨基酸侧链对蛋白的表达和组装有显著的影响。HPV11 L1蛋白的结构模建结果表明,aa364位于维系L1结构的重要β-折叠上,计算机突变分析提示aa364侧链参与的氢键形成以及与周围氨基酸的碰撞可能是影响VLP组装的原因。多HPV序列比对分析显示,相当于HPV11-L1 aa364的氨基酸在其它型别序列上高度保守,均为本研究所确认的Glu。最后,利用上述本研究的突变研究,选定HPV11N4C-L1(含Glu~(364))为目的基因进行HPV11预防性疫苗的研制。本研究成功地建立了一套发酵、纯化和复性工艺,并获得达到新药临床试验要求的HPV11型疫苗。动物实验结果显示,该疫苗可诱导小鼠产生较高滴度的中和抗体,中和抗体与HPV6型存在明显的交叉反应。总之,本研究为HPV11的L1蛋白性质研究、VLP组装机理提供了结构信息,为HPV11疫苗的分子设计提供了参考,成功地研制出针对HPV11的预防性疫苗,并为HPV6和HPV11杂合疫苗的研制提供了依据。
[Abstract]:Epidemiology and related studies have confirmed that HPV infection is closely related to condyloma acuminatum, cervical epitheliomatous hyperplasia and cervical cancer. HPV6 type 11 is the main pathogenic factor of condyloma acuminatum. About 90% of genital warts are related to HPV6 and HPV11. The development of vaccine to prevent HPV11 infection has good social and economic benefits. The results show that HPVL1 can be assembled independently to form HPV virus-like particles, thus the natural neutralizing epitopes of HPV can be reproduced well and have good immunogenicity, which is the main target protein of prophylactic vaccine research at present. In this study, the prokaryotic expression system was used to express HPV11 L1 protein and its mutants. The effects of N-terminal sequence and key amino acids on HPV11 L1 expression and VLP assembly efficiency were studied in order to obtain a vaccine for the prevention of HPV11 virus infection. First of all, the N-terminal deletion mutation of HPV11-L1 protein showed that the N-terminal deletion of HPV11-L1 protein did not affect the formation of 16aa, but had different effects on the expression of target protein. The mutant HPV11N4C-L1 with deletion of HPV11-L1 had a higher expression level in Escherichia coli. It can be used in the study of prophylactic vaccine. Secondly, the two HPV11 sequences in Genebank were found to be different in Aa364A453A501 (Glufus 364) and Lysnus 364Sequin453 and Tyrrhium 453 (Lysxanthine 501) and Arg501. the results showed that the physicochemical properties of the two proteins and the assembly of VLP were quite different from those of the two proteins. The results showed that there were significant differences between the two HPV11 sequences and that between the two HPV11 strains. The results showed that there were significant differences in the physical and chemical properties of the two proteins and the assembly of VLP. Site-directed mutagenesis test showed that the difference between A453 and aa501 did not affect the properties of the protein, but aa364 was very important to the assembly efficiency of VLP. Furthermore, the mutagenesis of aa364 into Glnsil-Asp-Argopheus gly and Leu showed that the side chain of amino acid had a significant effect on protein expression and assembly. The result of structural modeling of HPV11 L1 protein showed that Aa364 was located on the important 尾 -fold that holds the L1 structure. Computer mutation analysis suggested that the formation of hydrogen bonds in the side chain of aa364 and the collision with the surrounding amino acids might be the factors affecting the assembly of aa364. Multiple HPV-sequence alignment analysis showed that the amino acids corresponding to HPV11-L1 aa364 were highly conserved in other types, all of which were confirmed by our study. Finally, HPV11N4C-L1 was selected as the target gene for the preparation of HPV11 prophylactic vaccine. In this study, a set of fermentation, purification and renaturation processes were successfully established, and HPV11 vaccine was obtained to meet the requirements of new drug clinical trials. The results of animal experiments showed that the vaccine could induce higher titer of neutralizing antibody in mice, and there was obvious cross reaction between neutralizing antibody and HPV6 type. All in all, this study provides structural information for the study of L1 protein properties of HPV11 and provides a reference for the molecular design of HPV11 vaccine. A prophylactic vaccine against HPV11 was successfully developed. It also provides the basis for the development of hybrid vaccine of HPV6 and HPV11.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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