人乳头瘤病毒主要衣壳蛋白在大肠杆菌中可溶性表达的提升研究

发布时间：2018-05-02 04:30

本文选题：人乳头瘤病毒 + 主要衣壳蛋白　；参考：《吉林大学》2016年博士论文

【摘要】：人乳头瘤病毒(Human papillomavirus:HPV)是一类非包膜的双链DNA(ds DNA)病毒,能够侵染人体正常的皮肤和粘膜组织。HPV的感染将引起的皮肤、口腔、舌、扁桃体、咽喉、生殖器的良性或恶性病变,其中良性病变包括扁平疣、湿疣等,恶性病变则包括头颈癌、咽喉癌、宫颈癌、肛门癌等。据世界卫生组织调查,截止到2012年全球有1亿6千万宫颈癌临床病例,同年新增病例为52.8万并导致26.6万人的死亡,而我国每年有13万左右的新增病例,并有持续增长的趋势。通过对HPV导致宫颈上皮内瘤样病变(Cervical intraepithelial neoplasia:CIN)等级评估,采用电灼、激光、微波、冷冻、子宫颈电环切除术等方法,同时结合多种抗病毒药物(如重组人干扰素、阿普洛韦、白介素等)可进一步巩固治疗效果。但是由HPV引发的疾病难以有效控制,复发率较高,极大的增加了恶性肿瘤的发生几率。因此,对高危型HPV感染的预防成为人们关注的重点,其中最主要的手段为预防性疫苗的接种,可使接种者获得对HPV的预防能力。研究表明,由于HPV的中和抗原表位主要存在于主要衣壳蛋白L1的表面。因此,HPV类病毒颗粒(Virus-like particle:VLPs)可作为开展预防性疫苗研发的平台。到目前为止,已有三种获得美国食品药品监督管理局(U.S.Food and Drug Administration:FDA)许可的VLPs疫苗上市。虽然,HPV预防性疫苗并没有显著的宫颈癌治疗功能,而且其亚型限制以及高昂售价使得此类疫苗的推广、普及受到限制,尤其在宫颈癌高发的发展中国家中;但是,迄今为止预防性疫苗是预防高危型HPV感染的最为有效的药物并具有推广的可能性。因此,降低预防性疫苗的亚型限制以及有效的降低其生产成本是急需解决的问题。本论文的第一章我们对HPV的组成、生命周期、诱发的疾病、致病机理、筛查的手段、治疗方法、预防方法以及局限性进行了阐述,描述了热激蛋白的功能、作用机理以及在蛋白质折叠、可溶性表达的重要性,描述了HPV L1晶体结构以及helix 5对L1五聚体稳定性以及可溶性的影响,最后阐述本论文的立论依据以及研究意义。本论文的第二章为了解决HPV主要衣壳蛋白在大肠杆菌中可溶性表达量低的问题,我们通过构建与分子伴侣的共表达系统实现L1可溶性表达量的提升。在本章节中,主要衣壳蛋白以融合蛋白GST-L1的方式表达,以大肠杆菌为宿主分别与Gro EL/ES、Dna K/Dna J/Grp E或TF构建共表达系统。通过研究发现,在大肠杆菌中过量表达Gro EL/ES是增加GST-L1可溶性表达的主要因素。随着GST-L1可溶性表达量的提升,L1五聚体的产率也随之得到了明显的提升。同时,Gro EL/ES的过表达有效的提高了细胞的生长速率。在共表达系统中,由于Gro EL/ES的过量表达使得大量的GST-L1与其结合影响了L1的纯化以及五聚体的产率。因此,ATP与Mg Cl2引入纯化过程促进了GST-L1的释放,并提升了其与亲和层析柱的集合效率并最终促进了L1五聚体产率。通过对表达以及纯化条件的优化,使得HPV16、18、58 L1五聚体的产率得到明显的提升,分别是对照组的5、3以及2倍。重组HPV主要衣壳蛋白L1在大肠杆菌中的表达量的提升,为HPV预防性疫苗生产工艺的优化提供一种新方法。本论文的第三章我们基于HPV16 L1α螺旋5(helix5:h5)469位Leu残基(L469)的突变提升了L1五聚体的可溶性产率的现象,认为此位点与主要衣壳蛋白的自身稳定性相关。因此,将469位的Leu分别突变为Ala(A)、Gly(G)、Ser(S)、Lys(K)以观察L1五聚体可溶性产量的变化情况,并通过对突变体二级结构以及空间构象的分析探究影响L1五聚体可溶性产率的机制。同时,利用分子动力学模拟的手段对突变体的空间构象模型进行建立与分析,并对突变体中与h5相邻二级结构的变化进行分析,发现L469A中h5自身的螺旋以及空间上相邻的β折叠结构的变化使得L1的核心结构域更为紧密,同时有利于L1的稳定性以及表面亲水残基的暴露;而且L469A中h5柔性的增加影响了自身表面电荷的分布,有利于与界面残基的静电相互作用。这一系列变化有助于稳定L1的空间构象,同时促进了五聚体的形成与结构稳定性。
[Abstract]:Human papillomavirus (Human papillomavirus:HPV) is a non enveloped double stranded DNA (DS DNA) virus that can infect normal skin and mucous tissue of the human body, the mouth, tongue, tonsil, throat, and genitals of the skin, tongue, tonsil, pharynx, and genitals. Benign lesions include verruca flattened, condyloma, and malignant lesions including head Cervical cancer, laryngopharyngeal cancer, cervical cancer, anal cancer, etc.. According to the WHO survey, by 2012, there were 160 million clinical cases of cervical cancer in the world. 528 thousand new cases were added in the same year and resulted in the death of 266 thousand people. There were about 130 thousand new cases in China each year and the trend of continuous growth. By HPV, the cervical intraepithelial neoplasia was caused. Cervical intraepithelial neoplasia:CIN grade assessment, using electric cauterization, laser, microwave, cryopreservation, cervix resection and other methods, combined with a variety of antiviral drugs (such as recombinant human interferon, Apu Lowe, interleukins, etc.) can further consolidate the therapeutic effect. But the disease caused by HPV is difficult to be effectively controlled, the recurrence rate is high. It has greatly increased the incidence of malignant tumor. Therefore, the prevention of high risk HPV infection has become the focus of attention. The most important means is the vaccination of preventive vaccines, which can make the vaccinated people get the ability to prevent HPV. The study shows that the epitopes of the neutralization antigen of HPV mainly exist on the surface of the main capsid protein L1. In this case, the HPV virus particles (Virus-like particle:VLPs) can be used as a platform for developing preventive vaccine development. So far, there are three VLPs vaccines licensed by the US Food and Drug Administration (U.S.Food and Drug Administration:FDA). Subtype restrictions and high prices make the spread of such vaccines limited, especially in developing countries with high incidence of cervical cancer; however, so far preventive vaccines are the most effective drugs for preventing high risk HPV infections and are likely to be popularized. Therefore, subtype restrictions and effective reductions in preventive vaccines are therefore reduced. The first chapter of this paper is about the composition of the HPV, the life cycle, the disease, the pathogenesis, the means of screening, the method of treatment, the method of prevention, and the limitation, describing the function of the heat shock protein, the mechanism of action and the importance of the protein folding, the importance of soluble expression, and the importance of the expression of the protein. The structure of HPV L1 crystal and the effect of helix 5 on the stability and solubility of L1 five polymer are discussed. Finally, the basis and significance of this thesis are expounded. In order to solve the problem of low soluble expression of HPV main capsid protein in Escherichia coli, the second chapter of this paper is to construct a co expression system with a molecular chaperone. In this chapter, the main capsid protein is expressed in the way of fusion protein GST-L1, and the co expression system is constructed with Gro EL/ES, Dna K/Dna J/Grp E or TF, respectively, with Escherichia coli as the host. It is found that the overexpression of Gro EL/ ES in Escherichia coli is the main factor to increase the soluble expression of Gro. With the increase of GST-L1 soluble expression, the yield of L1 five polymer has also been greatly improved. Meanwhile, the overexpression of Gro EL/ES effectively improves the growth rate of cells. In co expression system, the excessive expression of Gro EL/ES makes a large number of GST-L1 and its binding influence the purification of L1 and the yield of the five polymer. Therefore, ATP The introduction of the purification process with Mg Cl2 promoted the release of GST-L1, enhanced the aggregation efficiency of the affinity chromatography column, and finally promoted the yield of L1 five polymer. By optimizing the expression and purification conditions, the yield of HPV16,18,58 L1 five polymer was obviously improved, respectively, the 5,3 of the control group and 2 times of the control group. The recombinant HPV main capsid eggs were reorganized. The enhancement of the expression of white L1 in Escherichia coli provides a new method for the optimization of the HPV prophylactic vaccine production process. In the third chapter of this paper, we improved the soluble yield of L1 five polymer based on the mutation of HPV16 L1 alpha helix 5 (helix5:h5) 469 Leu residues (L469), and considered the self stability of this site and the main capsid protein. Therefore, the 469 bits of Leu were mutated to Ala (A), Gly (G), Ser (S) and Lys (K) to observe the changes in the soluble yield of the L1 five polymer, and to explore the mechanism of the soluble yield of L1 five by the analysis of the structure of the two grade and the spatial conformation of the mutant, and the space of the mutant by the means of molecular dynamics simulation. The conformation model was established and analyzed, and the changes of the two adjacent structure of the adjacent H5 in the mutant were analyzed. It was found that the spiral of H5 itself and the change of the adjacent beta folding structure in the L469A made the core domain of the L1 more compact, at the same time, it was beneficial to the stability of L1 and the exposure of the surface hydrophilic residue, and the H5 flexibility in L469A. The increase affects the distribution of the surface charge, which is beneficial to the electrostatic interaction with the interface residues. This series of changes can help to stabilize the space conformation of L1 and promote the formation and structural stability of the five polymer.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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