本文選題：人乳頭瘤病毒 + 主要衣殼蛋白��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：人乳頭瘤病毒(Human papillomavirus:HPV)是一類(lèi)非包膜的雙鏈DNA(ds DNA)病毒,能夠侵染人體正常的皮膚和粘膜組織。HPV的感染將引起的皮膚、口腔、舌、扁桃體、咽喉、生殖器的良性或惡性病變,其中良性病變包括扁平疣、濕疣等,惡性病變則包括頭頸癌、咽喉癌、宮頸癌、肛門(mén)癌等。據(jù)世界衛(wèi)生組織調(diào)查,截止到2012年全球有1億6千萬(wàn)宮頸癌臨床病例,同年新增病例為52.8萬(wàn)并導(dǎo)致26.6萬(wàn)人的死亡,而我國(guó)每年有13萬(wàn)左右的新增病例,并有持續(xù)增長(zhǎng)的趨勢(shì)。通過(guò)對(duì)HPV導(dǎo)致宮頸上皮內(nèi)瘤樣病變(Cervical intraepithelial neoplasia:CIN)等級(jí)評(píng)估,采用電灼、激光、微波、冷凍、子宮頸電環(huán)切除術(shù)等方法,同時(shí)結(jié)合多種抗病毒藥物(如重組人干擾素、阿普洛韋、白介素等)可進(jìn)一步鞏固治療效果。但是由HPV引發(fā)的疾病難以有效控制,復(fù)發(fā)率較高,極大的增加了惡性腫瘤的發(fā)生幾率。因此,對(duì)高危型HPV感染的預(yù)防成為人們關(guān)注的重點(diǎn),其中最主要的手段為預(yù)防性疫苗的接種,可使接種者獲得對(duì)HPV的預(yù)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殼蛋白L1的表面。因此,HPV類(lèi)病毒顆粒(Virus-like particle:VLPs)可作為開(kāi)展預(yù)防性疫苗研發(fā)的平臺(tái)。到目前為止,已有三種獲得美國(guó)食品藥品監(jiān)督管理局(U.S.Food and Drug Administration:FDA)許可的VLPs疫苗上市。雖然,HPV預(yù)防性疫苗并沒(méi)有顯著的宮頸癌治療功能,而且其亞型限制以及高昂售價(jià)使得此類(lèi)疫苗的推廣、普及受到限制,尤其在宮頸癌高發(fā)的發(fā)展中國(guó)家中;但是,迄今為止預(yù)防性疫苗是預(yù)防高危型HPV感染的最為有效的藥物并具有推廣的可能性。因此,降低預(yù)防性疫苗的亞型限制以及有效的降低其生產(chǎn)成本是急需解決的問(wèn)題。本論文的第一章我們對(duì)HPV的組成、生命周期、誘發(fā)的疾病、致病機(jī)理、篩查的手段、治療方法、預(yù)防方法以及局限性進(jìn)行了闡述,描述了熱激蛋白的功能、作用機(jī)理以及在蛋白質(zhì)折疊、可溶性表達(dá)的重要性,描述了HPV L1晶體結(jié)構(gòu)以及helix 5對(duì)L1五聚體穩(wěn)定性以及可溶性的影響,最后闡述本論文的立論依據(jù)以及研究意義。本論文的第二章為了解決HPV主要衣殼蛋白在大腸桿菌中可溶性表達(dá)量低的問(wèn)題,我們通過(guò)構(gòu)建與分子伴侶的共表達(dá)系統(tǒng)實(shí)現(xiàn)L1可溶性表達(dá)量的提升。在本章節(jié)中,主要衣殼蛋白以融合蛋白GST-L1的方式表達(dá),以大腸桿菌為宿主分別與Gro EL/ES、Dna K/Dna J/Grp E或TF構(gòu)建共表達(dá)系統(tǒng)。通過(guò)研究發(fā)現(xiàn),在大腸桿菌中過(guò)量表達(dá)Gro EL/ES是增加GST-L1可溶性表達(dá)的主要因素。隨著GST-L1可溶性表達(dá)量的提升,L1五聚體的產(chǎn)率也隨之得到了明顯的提升。同時(shí),Gro EL/ES的過(guò)表達(dá)有效的提高了細(xì)胞的生長(zhǎng)速率。在共表達(dá)系統(tǒng)中,由于Gro EL/ES的過(guò)量表達(dá)使得大量的GST-L1與其結(jié)合影響了L1的純化以及五聚體的產(chǎn)率。因此,ATP與Mg Cl2引入純化過(guò)程促進(jìn)了GST-L1的釋放,并提升了其與親和層析柱的集合效率并最終促進(jìn)了L1五聚體產(chǎn)率。通過(guò)對(duì)表達(dá)以及純化條件的優(yōu)化,使得HPV16、18、58 L1五聚體的產(chǎn)率得到明顯的提升,分別是對(duì)照組的5、3以及2倍。重組HPV主要衣殼蛋白L1在大腸桿菌中的表達(dá)量的提升,為HPV預(yù)防性疫苗生產(chǎn)工藝的優(yōu)化提供一種新方法。本論文的第三章我們基于HPV16 L1α螺旋5(helix5:h5)469位Leu殘基(L469)的突變提升了L1五聚體的可溶性產(chǎn)率的現(xiàn)象,認(rèn)為此位點(diǎn)與主要衣殼蛋白的自身穩(wěn)定性相關(guān)。因此,將469位的Leu分別突變?yōu)锳la(A)、Gly(G)、Ser(S)、Lys(K)以觀察L1五聚體可溶性產(chǎn)量的變化情況,并通過(guò)對(duì)突變體二級(jí)結(jié)構(gòu)以及空間構(gòu)象的分析探究影響L1五聚體可溶性產(chǎn)率的機(jī)制。同時(shí),利用分子動(dòng)力學(xué)模擬的手段對(duì)突變體的空間構(gòu)象模型進(jìn)行建立與分析,并對(duì)突變體中與h5相鄰二級(jí)結(jié)構(gòu)的變化進(jìn)行分析,發(fā)現(xiàn)L469A中h5自身的螺旋以及空間上相鄰的β折疊結(jié)構(gòu)的變化使得L1的核心結(jié)構(gòu)域更為緊密,同時(shí)有利于L1的穩(wěn)定性以及表面親水殘基的暴露;而且L469A中h5柔性的增加影響了自身表面電荷的分布,有利于與界面殘基的靜電相互作用。這一系列變化有助于穩(wěn)定L1的空間構(gòu)象,同時(shí)促進(jìn)了五聚體的形成與結(jié)構(gòu)穩(wěn)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R392

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