HPV16L1蛋白的分子生物学研究

发布时间：2018-03-09 18:02

本文选题：人乳头瘤病毒L1蛋白　切入点：昆虫杆状病毒表达系统　出处：《西安交通大学》2006年博士论文　论文类型：学位论文

【摘要】： 【研究背景】人乳头瘤病毒(humanpapillomavirus,HPV) L1蛋白(major capsid protein)在HPV感染及其生活周期(life cycle)的各个阶段,特别是在病毒感染早期和诱发保护性体液免疫方面发挥重要作用。L1蛋白通过其羧基端的核定位信号(nuclear localization signal,NLS)先后二次进入宿主细胞核内,360分子的L1蛋白和12分子的L2蛋白通过二硫键组成的衣壳(capsid)包裹病毒DNA形成二十面体病毒毒粒(virion);散布于HPV衣壳表面的L1蛋白线性表位(linealepitopes)和构象表位(conformational epitopes)是诱发机体保护性免疫反应的主要抗原表位(antigen epitopes)。本论文主要针对L1蛋白的生物学特性进行初步研究,以期为阐明L1蛋白在HPV感染机制及其生活周期中的作用,研制HPV预防性疫苗提供理论依据和实验方法。【研究目的】 (1)利用生物信息学技术预测所有型别HPVs L1蛋白NLSs并予以分类,为HPV L1蛋白NLS及其核浆转运机制的研究提供指导。 (2)研究HPV16 L1蛋白在Sf-9细胞中入核转运动力学过程,探讨NLSHPV16L1(HPV16 L1 protein NLS)被用于靶向药物载体的可能性。 (3)研究不同亚细胞定位的表达产物对基因免疫诱导的机体体液免疫反应的影响。 (4)制备抗HPV16特异性L1IgY抗体(egg yolk immunoglobulin,IgY),为抗HPV16 L1蛋白抗体的制备和HPV16 L1蛋白的免疫检测探索新的方法。 (5)用噬菌体随机肽库技术(phage display random peptide library)筛选、分析HPV16 L1蛋白的抗原表位,为HPV16预防性疫苗的研制提供参考。【研究方法】 (1)从http://www.stdgen.lanl.gov/stdgen/virus , http://ca.expasy.org ,http://cubic.bioc.columbia.edu/predictNLS/和http://www.ncbi.nlm.nih.gov等数据库获取所有型HPVs L1蛋白氨基酸序列,利用PredictNLS软件对其进行NLS预测,根据经典NLS的一般规律及HPVs L1蛋白NLSs的同源性和结构特点予以分类。 (2)分别构建并经同源重组获得重组Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS、Ac-EGFP-NLSHPV16L1杆状病毒后,分别用其感染Sf-9昆虫细胞进行融合蛋白的表达,利用荧光显微镜和激光共聚焦显微镜观察增强型绿色荧光蛋白(enhanced green fluorescenceprotein,EGFP)标记的不同融合蛋白在Sf-9细胞内转运的动力学过程,分析NLSHPV16L1的核定位功能。 (3)构建重组pcDNA-EGFP-HPV16L1和pcDNA-EGFP-HPV16L1△NLS真核表达载体,以基因免疫的方式免疫BALB/c小鼠,检测血清抗EGFP特异性IgG抗体水平。 (4)利用由昆虫杆状病毒表达系统表达并经非变性法纯化的HPV16 L1蛋白免疫母鸡,制备抗HPV16 L1 IgY抗体。水稀释法纯化、ELISA法(Enzyme-linkedimmunosorbent assay)检测IgY抗体效价、免疫组织化学法(immunohistochemistry,IHC)和小鼠红细胞凝集抑制试验(hemagglutination inhibition assay,HAI)测定IgY抗体的活性。 (5)用抗HPV16 L1多克隆抗体筛选噬菌体随机肽库,获得阳性噬菌体克隆,并经免疫斑点法(immunity blot test)检测其与抗HPV16 L1多克隆抗体的免疫结合能力,经基因测序和同源性分析,确定HPV16 L1抗原表位。【结果】 (1)根据经典NLS的一般规律和HPVs L1蛋白NLSs的特征和组成,107型HPVs L1蛋白NLSs可分为15类,其中某些型别HPVs的L1蛋白含有与经典NLS不同的新的潜在NLS。 (2)分别用构建的重组Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS、Ac-EGFP、Ac-EGFP-NLSHPV16L1杆状病毒感染Sf-9细胞,进行融合蛋白的表达,发现EGFP均匀分布于Sf-9细胞内;包含NLSHPV16L1的EGFP-HPV16L1和EGFP-NLSHPV16L1融合蛋白主要积聚于Sf-9细胞核内;删除NLSHPV16L1的EGFP-HPV16L1△NLS融合蛋白滞留于Sf-9细胞浆内。此外,还获得了EGFP和EGFP-HPV16L1、EGFP-HPV16L1△NLS融合蛋白。 (3)重组pcDNA-EGFP-HPV16L1△NLS真核表达载体免疫组小鼠血清抗EGFP IgG抗体效价显著高于重组pcDNA-EGFP-HPV16L1真核表达载体免疫组小鼠血清抗EGFP IgG抗体效价(P0.001)。 (4)用HPV16 L1蛋白免疫母鸡制备的抗HPV16 L1IgY抗体可特异性与表达于CHO细胞内的EGFP-HPV16L1蛋白结合,并能抑制HPV16 L1病毒样颗粒(virus- like partical,VLP)介导的小鼠红细胞凝集(hemagglutination,HA)。 (5)用抗HPV16 L1多克隆抗体筛选噬菌体随机肽库并经同源性分析获得的5条多肽“TNLDLYG”、“IFDNHP”、“LTFKPQ”、“GIDS”、“NHGLLYSPLPT”可与抗HPV16 L1多克隆抗体发生免疫结合反应。【结论】 (1) 107型HPVs L1蛋白NLSs可分为15类,其中某些型别HPVs L1蛋白中具有与经典NLS不同的新的潜在NLS。这对于HPVs蛋白NLS和HPV L1蛋白核浆转运机制的研究具有一定的指导意义。 (2) pFB-EGFP昆虫杆状病毒转移载体的构建和EGFP的获得,为获取EGFP标记的融合蛋白、实现蛋白功能和相互作用的可视化研究提供了技术储备。 (3) HPV16 L1蛋白NLS具有核定位功能,有可能作为靶向药物载体。 (4) Sf-9细胞表达的EGFP-HPV16L1和EGFP-HPV16L1△NLS是双功能融合蛋白,该双功能融合蛋白的制备为HPV16感染机制和L1蛋白生物学行为的可视化研究提供了可能。 (5)在HPV16 L1蛋白氨基端融合较长基因或删除其羧基端的NLS均不影响VLP的形成。 (6)表达于细胞浆内的蛋白较细胞核内蛋白能诱发更高的经基因免疫途径介导的机体体液免疫应答水平。 (7)抗HPV16 L1 IgY抗体的制备，，为大量制备抗HPV16 L1蛋白抗体和HPV16 L1蛋白的免疫检测提供了新的方法。 (8)“TNLDLYG”、“IFDNHP”、“LTFKPQ”、“GIDS”、“NHGLLYSPLPT”多肽可能与HPV16 L1蛋白构象表位有关。
[Abstract]:[research background]
Human papilloma virus (humanpapillomavirus, HPV) L1 protein (major capsid protein) in HPV infection and its life cycle (life cycle) of each stage, especially play an important role of.L1 protein nuclear localization signal through its carboxyl terminal in the early stage of infection and induce protective humoral immunity (nuclear localization, signal, NLS) has two times into the host cell nucleus, 360 molecules of L1 protein and 12 molecules of L2 protein composition by two disulfide bonds of the capsid (capsid) package form twenty surface body virus DNA virus particles (virion); in HPV capsid L1 protein epitopes (linealepitopes) and conformational epitopes (conformational epitope) is the main antigen epitope induce protective immune responses (antigen epitopes) were studied in this paper. According to the biological characteristics of L1 protein, in order to clarify the L1 protein in HPV infected machine The role of the system and its life cycle and the development of the HPV preventive vaccine provide theoretical basis and experimental methods.
[purpose]
(1) the use of bioinformatics to predict all types of HPVs L1 protein NLSs and to provide guidance for the research of HPV classification, L1 protein NLS and its nucleocytoplasmic transport mechanism.
(2) to study the movement of HPV16 L1 protein in Sf-9 cells, and to explore the possibility of NLSHPV16L1 (HPV16 L1 protein NLS) being used as a target drug carrier.
(3) to study the effect of the expression products of different subcellular localization on the body humoral immune response induced by gene immunization.
(4) prepare anti HPV16 specific L1IgY egg (yolk immunoglobulin, IgY), and explore new ways for the preparation of antibodies against HPV16 L1 protein and the detection of HPV16 L1 protein.
(5) using phage random peptide library technology (phage display random peptide library) to screen and analyze the epitopes of HPV16 L1 protein, so as to provide references for the development of HPV16 preventive vaccine.
[research methods]
(1) from http://www.stdgen.lanl.gov/stdgen/virus, http://ca.expasy.org, http://cubic.bioc.columbia.edu/predictNLS/ and http://www.ncbi.nlm.nih.gov database for all type HPVs amino acid sequence of L1 protein, NLS prediction based on the PredictNLS software, to be classified according to the homology and structure characteristics of the general rules of classical NLS and HPVs L1 protein NLSs.
(2) were constructed by homologous recombination to obtain the recombinant Ac-EGFP, Ac-EGFP-HPV16L1, Ac-EGFP-HPV16L1, NLS, Ac-EGFP-NLSHPV16L1 respectively with the baculovirus after infected Sf-9 insect cells to express the fusion protein by fluorescence microscopy and laser confocal microscopy observation of enhanced green fluorescent protein (enhanced green, fluorescenceprotein, EGFP) of different transport kinetics of fusion protein markers in Sf-9 cells, analysis of nuclear localization of NLSHPV16L1.
(3) construction of recombinant pcDNA-EGFP-HPV16L1 and pcDNA-EGFP-HPV16L1 Delta NLS eukaryotic expression vector by gene immunization of immune BALB/c mice, anti EGFP serum specific IgG antibody levels.
(4) the expression of L1 protein by HPV16 system and immune purification method by non denatured hen baculovirus expression and preparation of anti HPV16 antibody L1 IgY. Purified water dilution method, ELISA method (Enzyme-linkedimmunosorbent assay) to detect IgY antibody titer, immunohistochemistry (immunohistochemistry, IHC) and hemagglutination inhibition test in mice (hemagglutination inhibition assay, HAI) for the determination of IgY antibody activity.
(5) HPV16 with anti L1 polyclonal antibody, phage displayed random peptide library, positive phage clones were obtained, and by Dot-ELISA (immunity blot test) and anti HPV16 L1 polyclonal antibody binding ability of immune detection, DNA sequencing and homology analysis, determine the HPV16 L1 epitope.
[results]
(1) according to the general rule of classic NLS and the characteristics and composition of HPVs L1 protein NLSs, type 107 HPVs L1 protein NLSs can be divided into 15 categories. Among them, L1 protein of some types HPVs contains new potential NLS. different from classic NLS.
(2) were used to construct the recombinant Ac-EGFP-HPV16L1, Ac-EGFP-HPV16L1 NLS, Ac-EGFP, Ac-EGFP-NLSHPV16L1 in baculovirus infected Sf-9 cells, expression of fusion protein, found that the uniform distribution of EGFP in Sf-9 cell; EGFP-HPV16L1 and EGFP-NLSHPV16L1 containing NLSHPV16L1 fusion protein mainly accumulates in the nucleus of Sf-9 cells; EGFP-HPV16L1 Delta NLS deletion of NLSHPV16L1 fusion protein on retention Sf-9 cytoplasm. In addition, also received EGFP and EGFP-HPV16L1, EGFP-HPV16L1 a NLS fusion protein.
(3) anti EGFP vector immunized mice serum IgG antibody titer was significantly higher than that of anti EGFP IgG antibody titer of immunized mice sera carrier eukaryotic expression recombinant pcDNA-EGFP-HPV16L1 eukaryotic expression recombinant pcDNA-EGFP-HPV16L1 NLS (P0.001).
(4) the expression of L1 protein and HPV16 in hens vaccinated against HPV16 was prepared by L1IgY antibody in CHO cells EGFP-HPV16L1 protein binding, and can inhibit the HPV16 L1 virus like particles (virus- like, partical, VLP) mice erythrocyte agglutination mediated (hemagglutination, HA).
(5) using the anti HPV16 L1 polyclonal antibody to screen phage random peptide library and homology analysis, we obtained 5 peptides, "TNLDLYG", "IFDNHP", "LTFKPQ", "GIDS" and "NHGLLYSPLPT", which could react with HPV16 antibody against polyclonal antibody.
[Conclusion]
(1) 107 HPVs L1 protein NLSs can be divided into 15 categories, some of which type HPVs L1 protein has certain guiding significance different from the classical NLS NLS. for this new potential HPVs protein NLS and HPV protein L1 nucleocytoplasmic transport mechanism.
(2) the construction of pFB-EGFP insect baculovirus transfer vector and the acquisition of EGFP provide a technological reserve for obtaining EGFP labeled fusion protein and visualizing protein function and interaction.
(3) HPV16 L1 protein NLS has nuclear location function and may be used as a target drug carrier.
(4) the expression of Sf-9 EGFP-HPV16L1 and EGFP-HPV16L1 NLS is a bifunctional fusion protein, may provide the bifunctional fusion protein were prepared for the study of visual mechanisms of infection and L1 protein in the biological behavior of HPV16.
(5) the formation of VLP is not affected by the fusion of the longer gene or the deletion of the NLS of the carboxyl terminus at the HPV16 L1 protein amino terminal.
(6) the protein expressed in the cytoplasm can induce a higher level of humoral immune response in the body, which is mediated by the gene immunization pathway.
(7) the preparation of anti HPV16 L1 IgY antibody provides a new method for the preparation of a large number of immunoassays for the preparation of anti HPV16 L1 protein antibody and HPV16 L1 protein.
(8) "TNLDLYG", "IFDNHP", "LTFKPQ", "GIDS", "NHGLLYSPLPT" polypeptide may be related to the conformation epitopes of the HPV16 L1 protein.

【学位授予单位】：西安交通大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R373

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