本文選題：人乳頭瘤病毒L1蛋白　切入點(diǎn)：昆蟲桿狀病毒表達(dá)系統(tǒng)　出處：《西安交通大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】： 【研究背景】 人乳頭瘤病毒(humanpapillomavirus,HPV) L1蛋白(major capsid protein)在HPV感染及其生活周期(life cycle)的各個(gè)階段,特別是在病毒感染早期和誘發(fā)保護(hù)性體液免疫方面發(fā)揮重要作用。L1蛋白通過其羧基端的核定位信號(hào)(nuclear localization signal,NLS)先后二次進(jìn)入宿主細(xì)胞核內(nèi),360分子的L1蛋白和12分子的L2蛋白通過二硫鍵組成的衣殼(capsid)包裹病毒DNA形成二十面體病毒毒粒(virion);散布于HPV衣殼表面的L1蛋白線性表位(linealepitopes)和構(gòu)象表位(conformational epitopes)是誘發(fā)機(jī)體保護(hù)性免疫反應(yīng)的主要抗原表位(antigen epitopes)。本論文主要針對(duì)L1蛋白的生物學(xué)特性進(jìn)行初步研究,以期為闡明L1蛋白在HPV感染機(jī)制及其生活周期中的作用,研制HPV預(yù)防性疫苗提供理論依據(jù)和實(shí)驗(yàn)方法。 【研究目的】 (1)利用生物信息學(xué)技術(shù)預(yù)測(cè)所有型別HPVs L1蛋白NLSs并予以分類,為HPV L1蛋白NLS及其核漿轉(zhuǎn)運(yùn)機(jī)制的研究提供指導(dǎo)。 (2)研究HPV16 L1蛋白在Sf-9細(xì)胞中入核轉(zhuǎn)運(yùn)動(dòng)力學(xué)過程,探討NLSHPV16L1(HPV16 L1 protein NLS)被用于靶向藥物載體的可能性。 (3)研究不同亞細(xì)胞定位的表達(dá)產(chǎn)物對(duì)基因免疫誘導(dǎo)的機(jī)體體液免疫反應(yīng)的影響。 (4)制備抗HPV16特異性L1IgY抗體(egg yolk immunoglobulin,IgY),為抗HPV16 L1蛋白抗體的制備和HPV16 L1蛋白的免疫檢測(cè)探索新的方法。 (5)用噬菌體隨機(jī)肽庫(kù)技術(shù)(phage display random peptide library)篩選、分析HPV16 L1蛋白的抗原表位,為HPV16預(yù)防性疫苗的研制提供參考。 【研究方法】 (1)從http://www.stdgen.lanl.gov/stdgen/virus , http://ca.expasy.org ,http://cubic.bioc.columbia.edu/predictNLS/和http://www.ncbi.nlm.nih.gov等數(shù)據(jù)庫(kù)獲取所有型HPVs L1蛋白氨基酸序列,利用PredictNLS軟件對(duì)其進(jìn)行NLS預(yù)測(cè),根據(jù)經(jīng)典NLS的一般規(guī)律及HPVs L1蛋白NLSs的同源性和結(jié)構(gòu)特點(diǎn)予以分類。 (2)分別構(gòu)建并經(jīng)同源重組獲得重組Ac-EGFP、Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS、Ac-EGFP-NLSHPV16L1桿狀病毒后,分別用其感染Sf-9昆蟲細(xì)胞進(jìn)行融合蛋白的表達(dá),利用熒光顯微鏡和激光共聚焦顯微鏡觀察增強(qiáng)型綠色熒光蛋白(enhanced green fluorescenceprotein,EGFP)標(biāo)記的不同融合蛋白在Sf-9細(xì)胞內(nèi)轉(zhuǎn)運(yùn)的動(dòng)力學(xué)過程,分析NLSHPV16L1的核定位功能。 (3)構(gòu)建重組pcDNA-EGFP-HPV16L1和pcDNA-EGFP-HPV16L1△NLS真核表達(dá)載體,以基因免疫的方式免疫BALB/c小鼠,檢測(cè)血清抗EGFP特異性IgG抗體水平。 (4)利用由昆蟲桿狀病毒表達(dá)系統(tǒng)表達(dá)并經(jīng)非變性法純化的HPV16 L1蛋白免疫母雞,制備抗HPV16 L1 IgY抗體。水稀釋法純化、ELISA法(Enzyme-linkedimmunosorbent assay)檢測(cè)IgY抗體效價(jià)、免疫組織化學(xué)法(immunohistochemistry,IHC)和小鼠紅細(xì)胞凝集抑制試驗(yàn)(hemagglutination inhibition assay,HAI)測(cè)定IgY抗體的活性。 (5)用抗HPV16 L1多克隆抗體篩選噬菌體隨機(jī)肽庫(kù),獲得陽性噬菌體克隆,并經(jīng)免疫斑點(diǎn)法(immunity blot test)檢測(cè)其與抗HPV16 L1多克隆抗體的免疫結(jié)合能力,經(jīng)基因測(cè)序和同源性分析,確定HPV16 L1抗原表位。 【結(jié)果】 (1)根據(jù)經(jīng)典NLS的一般規(guī)律和HPVs L1蛋白NLSs的特征和組成,107型HPVs L1蛋白NLSs可分為15類,其中某些型別HPVs的L1蛋白含有與經(jīng)典NLS不同的新的潛在NLS。 (2)分別用構(gòu)建的重組Ac-EGFP-HPV16L1、Ac-EGFP-HPV16L1△NLS、Ac-EGFP、Ac-EGFP-NLSHPV16L1桿狀病毒感染Sf-9細(xì)胞,進(jìn)行融合蛋白的表達(dá),發(fā)現(xiàn)EGFP均勻分布于Sf-9細(xì)胞內(nèi);包含NLSHPV16L1的EGFP-HPV16L1和EGFP-NLSHPV16L1融合蛋白主要積聚于Sf-9細(xì)胞核內(nèi);刪除NLSHPV16L1的EGFP-HPV16L1△NLS融合蛋白滯留于Sf-9細(xì)胞漿內(nèi)。此外,還獲得了EGFP和EGFP-HPV16L1、EGFP-HPV16L1△NLS融合蛋白。 (3)重組pcDNA-EGFP-HPV16L1△NLS真核表達(dá)載體免疫組小鼠血清抗EGFP IgG抗體效價(jià)顯著高于重組pcDNA-EGFP-HPV16L1真核表達(dá)載體免疫組小鼠血清抗EGFP IgG抗體效價(jià)(P0.001)。 (4)用HPV16 L1蛋白免疫母雞制備的抗HPV16 L1IgY抗體可特異性與表達(dá)于CHO細(xì)胞內(nèi)的EGFP-HPV16L1蛋白結(jié)合,并能抑制HPV16 L1病毒樣顆粒(virus- like partical,VLP)介導(dǎo)的小鼠紅細(xì)胞凝集(hemagglutination,HA)。 (5)用抗HPV16 L1多克隆抗體篩選噬菌體隨機(jī)肽庫(kù)并經(jīng)同源性分析獲得的5條多肽“TNLDLYG”、“IFDNHP”、“LTFKPQ”、“GIDS”、“NHGLLYSPLPT”可與抗HPV16 L1多克隆抗體發(fā)生免疫結(jié)合反應(yīng)。 【結(jié)論】 (1) 107型HPVs L1蛋白NLSs可分為15類,其中某些型別HPVs L1蛋白中具有與經(jīng)典NLS不同的新的潛在NLS。這對(duì)于HPVs蛋白NLS和HPV L1蛋白核漿轉(zhuǎn)運(yùn)機(jī)制的研究具有一定的指導(dǎo)意義。 (2) pFB-EGFP昆蟲桿狀病毒轉(zhuǎn)移載體的構(gòu)建和EGFP的獲得,為獲取EGFP標(biāo)記的融合蛋白、實(shí)現(xiàn)蛋白功能和相互作用的可視化研究提供了技術(shù)儲(chǔ)備。 (3) HPV16 L1蛋白NLS具有核定位功能,有可能作為靶向藥物載體。 (4) Sf-9細(xì)胞表達(dá)的EGFP-HPV16L1和EGFP-HPV16L1△NLS是雙功能融合蛋白,該雙功能融合蛋白的制備為HPV16感染機(jī)制和L1蛋白生物學(xué)行為的可視化研究提供了可能。 (5)在HPV16 L1蛋白氨基端融合較長(zhǎng)基因或刪除其羧基端的NLS均不影響VLP的形成。 (6)表達(dá)于細(xì)胞漿內(nèi)的蛋白較細(xì)胞核內(nèi)蛋白能誘發(fā)更高的經(jīng)基因免疫途徑介導(dǎo)的機(jī)體體液免疫應(yīng)答水平。 (7)抗HPV16 L1 IgY抗體的制備，，為大量制備抗HPV16 L1蛋白抗體和HPV16 L1蛋白的免疫檢測(cè)提供了新的方法。 (8)“TNLDLYG”、“IFDNHP”、“LTFKPQ”、“GIDS”、“NHGLLYSPLPT”多肽可能與HPV16 L1蛋白構(gòu)象表位有關(guān)。
[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.

【學(xué)位授予單位】：西安交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 陳宏偉,鄭瑾,楊筱鳳,王靜,來寶長(zhǎng),司履生,王一理;利用His-桿狀病毒表達(dá)系統(tǒng)制備HPV16L1VLP[J];西北大學(xué)學(xué)報(bào)(自然科學(xué)版);2005年01期

2 鄭濱,王健偉,姜惠英,屈建國(guó),王一禮,司履生,董小平;利用昆蟲—桿狀病毒表達(dá)系統(tǒng)表達(dá)人乳頭瘤病毒16型L1蛋白[J];中華實(shí)驗(yàn)和臨床病毒學(xué)雜志;2001年04期

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