本文選題：登革病毒 + E蛋白�。� 參考：《第二軍醫(yī)大學(xué)》2011年博士論文

【摘要】：登革病毒是黃病毒屬、有包膜的單正鏈RNA病毒,根據(jù)其包膜的抗原性不同,分為四種血清型(DENV1-4),主要以埃及伊蚊和白紋伊蚊為傳播媒介,廣泛流行于熱帶和亞熱帶地區(qū)。臨床上主要引起登革熱(Dengue Fever,DF)、登革出血熱(Dengue hemorrhagic fever,DHF)和登革休克綜合征(dengue shock syndrome,DSS)。其中,DF是自限性發(fā)熱性疾病;而DHF/DSS則引發(fā)血管通透性顯著增加,導(dǎo)致血漿滲漏引發(fā)休克,嚴(yán)重威脅患者的生命。世界上約有半數(shù)的人口生活在登革熱疫區(qū),每年有超過(guò)5000萬(wàn)的感染病例,其中有50萬(wàn)人發(fā)展為嚴(yán)重的登革出血熱和登革休克綜合征。但是,臨床上缺乏預(yù)防和治療DENV感染的有效疫苗和藥物,主要以對(duì)癥支持治療為主�；谂R床上針對(duì)DENV感染無(wú)有效防治手段的現(xiàn)狀,治療性抗體策略獲得了廣泛關(guān)注。大量體內(nèi)、外實(shí)驗(yàn)證明:中和抗體能有效阻止DENV的感染,不僅能發(fā)揮病毒感染前的預(yù)防作用,而且在病毒感染后的一段時(shí)期內(nèi),依然能發(fā)揮治療病毒感染的作用。 DENV基因組編碼3個(gè)結(jié)構(gòu)蛋白(核蛋白C,膜結(jié)合蛋白M和包膜蛋白E)和7種非結(jié)構(gòu)蛋白( NS1、NS2a、NS2b、NS3、NS4a、NS4b和NS5)。其中,包膜E蛋白是病毒的主要結(jié)構(gòu)蛋白,位于成熟病毒顆粒表面,排列成平行二聚體結(jié)構(gòu),構(gòu)成病毒顆粒的主要突起。E蛋白在空間結(jié)構(gòu)上形成3個(gè)不同的結(jié)構(gòu)域(ED1,ED2和ED3),其中,第3個(gè)結(jié)構(gòu)域(ED3)介導(dǎo)了病毒與靶細(xì)胞的吸附,包含最重要的中和表位。 但是,E蛋白上中和性表位知之甚少,同一血清型登革病毒內(nèi)不同基因型病毒株之間的差異對(duì)抗體保護(hù)作用的影響也還有待于進(jìn)一步分析。這些問(wèn)題都阻礙了登革病毒致病機(jī)理的闡明及防治手段的開(kāi)發(fā)。因此,確定登革病毒E蛋白的中和性表位,闡明抗體中和作用的機(jī)制,進(jìn)一步探討病毒致病性相關(guān)的關(guān)鍵氨基酸殘基,不僅能促進(jìn)對(duì)其致病機(jī)制的了解,也為登革熱的防治提供重要信息;無(wú)論從DENV發(fā)病機(jī)理,還是研發(fā)疫苗和治療藥物,都具有重要意義。 在登革病毒致病機(jī)制及其抗體治療策略的研究過(guò)程中,人們發(fā)現(xiàn)交叉反應(yīng)性非中和抗體以及亞中和濃度的抗體能夠通過(guò)抗體的Fc段產(chǎn)生抗體依賴的感染增強(qiáng)作用(ADE),進(jìn)而促進(jìn)疾病的進(jìn)展,這種現(xiàn)象阻礙了疫苗和單克隆抗體在登革熱及其他相關(guān)病毒防治中的應(yīng)用。登革熱常發(fā)生幾種血清型的交叉感染,因此,抗體要應(yīng)用于治療,必須能同時(shí)對(duì)四種血清型病毒均能產(chǎn)生有效的保護(hù)�，F(xiàn)有的強(qiáng)中和活性的單克隆抗體,多為單個(gè)型或只針對(duì)少數(shù)幾個(gè)型的單克隆抗體,雖也有多株交叉反應(yīng)性抗體,但針對(duì)不同血清型病毒的保護(hù)效價(jià)不一;而幾種抗體的混合應(yīng)用又存在成分復(fù)雜以及需進(jìn)行多次臨床試驗(yàn)的問(wèn)題。雙可變區(qū)抗體技術(shù)( Dual-Variable-DomainImmunoglobulin, DVD-Ig)可以通過(guò)將2株不同抗體可變區(qū)構(gòu)建于一株抗體分子上并保持原有抗體的親和力和保護(hù)活性,大大簡(jiǎn)化了多株抗體混合應(yīng)用帶來(lái)的麻煩;獲得的抗體可進(jìn)一步經(jīng)過(guò)基因工程改造,將抗體的Fc斷改造以消除ADE。在針對(duì)四種血清型登革病毒的抗體治療過(guò)程中,通過(guò)將只針對(duì)幾個(gè)型的具有強(qiáng)中和活性的單克隆抗體進(jìn)行重新構(gòu)建,可獲得同時(shí)針對(duì)四種血清型登革病毒具有廣譜中和活性的雙可變區(qū)抗體。 本項(xiàng)研究旨在利用雜交瘤篩選技術(shù)篩選獲得具有中和活性的單克隆抗體,驗(yàn)證其體內(nèi)外中和活性,進(jìn)一步分析登革病毒ED3蛋白的中和表位,明確了抗體結(jié)合的關(guān)鍵位點(diǎn),初步闡明其中和作用的機(jī)制;進(jìn)一步采用基因工程方法構(gòu)建嵌合抗體及無(wú)ADE活性的廣譜中和性抗體。研究主要包括四個(gè)部分: 一、登革病毒ED3區(qū)特異單克隆抗體的制備及功能鑒定 我們以重組串聯(lián)的四種血清型登革病毒ED3蛋白作為免疫原,采用常規(guī)方法建立分泌針對(duì)登革病毒ED3區(qū)的雜交瘤細(xì)胞系。最終獲得了14株登革病毒ED3區(qū)特異單抗(1B12、1E12、1G6、1H8、2F9、2G9、2H12、3A10、3H12、4H10、5C10、6E1、6H7和7G5)。 為了對(duì)這14株單抗所針對(duì)的病毒進(jìn)行鑒定,我們以四種血清型病毒感染的BHK細(xì)胞制備的抗原片對(duì)其進(jìn)行免疫熒光檢測(cè)。結(jié)果證實(shí),這14株雜交瘤細(xì)胞所分泌的單抗均特異性針對(duì)登革病毒,對(duì)其他黃病毒屬成員不具有交叉反應(yīng);14株單抗對(duì)不同血清型登革病毒的交叉反應(yīng)測(cè)定結(jié)果顯示,1B12、1E12、2H12、3A10、5C10、6H7和7G5是登革1型病毒特異的單抗;1H8和2G9為登革3型病毒特異單抗;1G6為登革4型病毒特異單抗;2F9可交叉結(jié)合登革1、3型病毒;3H12和4H10可交叉結(jié)合1-3型登革病毒;6E1可交叉結(jié)合四種血清型病毒。上述結(jié)果表明,我們獲得了ED3結(jié)構(gòu)域特異的針對(duì)四種血清型登革病毒的單抗,為ED3蛋白的抗原表位分析、登革病毒的診斷以及新型疫苗和抗病毒藥物研究提供重要信息和工具。 采用蝕斑中和減少試驗(yàn)檢測(cè)這14株登革病毒特異單抗的體外中和活性,獲得一株登革4型病毒特異的中和抗體;其次,對(duì)體外有中和活性的單抗采用乳鼠顱內(nèi)保護(hù)模型觀察其體內(nèi)保護(hù)作用。結(jié)果顯示,其中一株特異性針對(duì)登革4型病毒的單抗1G6具有較強(qiáng)的體外中和活性和體內(nèi)保護(hù)作用,單一50μg劑量的抗體可以可在登革4型病毒感染4h和24h后分別使90%和30%的小鼠存活,表明它對(duì)登革病毒的感染具有一定治療作用。 為了確定中和抗體的作用機(jī)制,我們采用吸附前后蝕斑實(shí)驗(yàn)測(cè)定其在病毒感染周期中發(fā)揮作用的方式。結(jié)果顯示,1G6主要通過(guò)抑制登革病毒與靶細(xì)胞的吸附來(lái)發(fā)揮中和作用;當(dāng)病毒與靶細(xì)胞吸附之后,其中和效價(jià)大大降低。 二、中和表位的篩選與鑒定 為了分析登革4型病毒特異性中和抗體的中和表位,我們首先采用噬菌體隨機(jī)12肽庫(kù)對(duì)單抗1G6抗原表位進(jìn)行篩選獲得了其一致表位序列,并進(jìn)行了每5個(gè)氨基酸的系列缺失突變進(jìn)一步證實(shí)了肽庫(kù)的篩選結(jié)果。序列比對(duì)結(jié)果顯示,該單抗表位所對(duì)應(yīng)的多肽序列位于登革4型病毒E蛋白第3結(jié)構(gòu)域387LTLH390區(qū)域。在一級(jí)結(jié)構(gòu)上,登革4型病毒E蛋白第388和390位氨基酸與登革1-3型病毒ED3蛋白存在不同;從已有結(jié)晶的三維結(jié)構(gòu)上分析,發(fā)現(xiàn),位于第390位的組氨酸,含有一個(gè)與其它型登革病毒所不同的咪唑環(huán)結(jié)構(gòu)。 為了確定單抗1G6與E蛋白結(jié)合的關(guān)鍵位點(diǎn),我們進(jìn)一步進(jìn)行了定點(diǎn)突變并以間接ELISA方式測(cè)定不同氨基酸位點(diǎn)的突變對(duì)抗體結(jié)合力的影響。結(jié)果顯示,當(dāng)T388G和H390G單獨(dú)突變時(shí),單抗1G6與ED3蛋白的結(jié)合力有顯著下降;當(dāng)二者聯(lián)合突變時(shí),其結(jié)合力完全喪失,表明登革病毒E蛋白T388和H390位是1G6與其結(jié)合的關(guān)鍵位點(diǎn)。 三、嵌合抗體的構(gòu)建及其生物學(xué)特性 為了減弱鼠源抗體的異源反應(yīng)及下一步構(gòu)建具有廣譜中和活性的雙可變區(qū)抗體,首先用5’RACE法獲得了鼠源抗體1G6的輕重鏈可變區(qū)序列,并全基因合成了具有交叉中和登革1-3型病毒的抗體1A1D-2的輕重鏈可變區(qū)序列。在此基礎(chǔ)上,將輕重鏈可變區(qū)分別與人重鏈(IgG1)和輕鏈(κ)恒定區(qū)連接構(gòu)成全長(zhǎng)人-鼠嵌合抗體后分別裝入真核表達(dá)載體pcDNA3.1(+),構(gòu)建了嵌合抗體輕重鏈的表達(dá)載體,共轉(zhuǎn)染CHO細(xì)胞,經(jīng)G418壓力篩選獲得可穩(wěn)定分泌人-鼠嵌合抗體的穩(wěn)轉(zhuǎn)細(xì)胞株。實(shí)驗(yàn)證明,該抗體具有與親本鼠源抗體相同的抗原結(jié)合活性和體外中和活性,為下一步構(gòu)建雙可變區(qū)中和抗體奠定了基礎(chǔ)。 四、無(wú)ADE活性的雙可變區(qū)抗體的構(gòu)建及功能鑒定 采用基因工程技術(shù),以overlap PCR的方式將單抗1A1D-2和1G6的輕重鏈可變區(qū)序列分別以9個(gè)氨基酸的linker進(jìn)行前后連接,構(gòu)建成DVD1A1D-1G6-VL、DVD1G6-1A1D-VL和DVD1A1D-1G6-VH、DVD1G6-1A1D-VH片段,分別與人IgG 1的輕重鏈恒定區(qū)連接后裝入表達(dá)載體表達(dá)載體pcDNA3.1(+),共轉(zhuǎn)染CHO細(xì)胞,G418篩選獲得2種結(jié)構(gòu)的DVD抗體穩(wěn)轉(zhuǎn)細(xì)胞株,無(wú)血清大量培養(yǎng)及抗體純化,體外結(jié)合實(shí)驗(yàn)證實(shí),DVD1A1D-1G6的結(jié)合活性要優(yōu)于DVD1G6-1A1D。體內(nèi)外實(shí)驗(yàn)證明,DVD1A1D-1G6抗體保持了雙價(jià)抗體的體內(nèi)外中和活性,實(shí)現(xiàn)了抗病毒譜的擴(kuò)大,具有抗四種血清型登革病毒活性。進(jìn)一步將介導(dǎo)ADE效應(yīng)的Fc段缺失突變后,其ADE活性消失,同時(shí)Fc段的突變對(duì)其中和活性無(wú)明顯影響,表明我們成功構(gòu)建了具有廣譜抗登革病毒的無(wú)ADE中和抗體。 本研究篩選獲得了14株登革病毒特異單克隆抗體,其中一株新型登革4型病毒特異性中和抗體,發(fā)現(xiàn)了新的E蛋白功能表位,為闡明E蛋白的結(jié)構(gòu)與功能、登革病毒致病和免疫的分子機(jī)理奠定了理論基礎(chǔ);進(jìn)一步構(gòu)建獲得了無(wú)ADE活性的可同時(shí)中和四種血清型登革病毒的雙可變區(qū)抗體,為研制抗登革病毒新型抗體藥物作出了探索和提供了重要信息。
[Abstract]:Dengue virus (dengue virus) is the genus of the genus yellows, with coated single positive chain RNA virus. According to its membrane antigenicity, it is divided into four serotypes (DENV1-4). It is mainly used as the medium of Aedes aegypti and Aedes albopictus, widely prevalent in the tropics and subtropics. It mainly causes Dengue Fever (DF) and dengue haemorrhagic fever (Dengue hemorrhagic). Fever, DHF) and dengue shock syndrome (dengue shock syndrome, DSS). Among them, DF is a self limiting febrile disease, and DHF/DSS causes a significant increase in vascular permeability, resulting in shock caused by plasma leakage and a serious threat to the lives of patients. About half of the population of the world lives in dengue fever epidemic areas, with more than 50 million cases of infection each year, 500 thousand of them developed into severe dengue hemorrhagic fever and dengue shock syndrome. However, the clinical lack of effective vaccines and drugs for the prevention and treatment of DENV infection is mainly due to symptomatic support therapy. Based on the clinical status of the non effective prevention and treatment of DENV infection, the therapeutic antibody strategy has received extensive attention. The experiments have proved that neutralizing antibodies can effectively prevent DENV infection, not only play a preventive role before the virus infection, but also play a role in the treatment of virus infection in a period after the virus infection.
The DENV genome encodes 3 structural proteins (nuclear protein C, membrane binding protein M and envelope protein E) and 7 non structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). Among them, the envelope protein is the main structural protein of the virus, located on the surface of the mature virus particles and arranged in a parallel two polymer structure, which constitutes the main protuberance of the virus particles in the air. 3 different domains (ED1, ED2 and ED3) are formed in the inter structure, of which third domains (ED3) mediate the adsorption of the virus and the target cells, including the most important neutralization epitopes.
However, little is known about the neutralization epitopes on the E protein, and the impact of different genotypes in the same serotype dengue virus on the protective effect of the antibody still remains to be further analyzed. These problems have hindered the elucidation of the pathogenesis of dengue virus and the development of the means of prevention and control. Therefore, the neutralization of the E protein of dengue virus is determined. The sexual epitopes, which elucidate the mechanism of antibody neutralization, further explore the key amino acid residues associated with viral pathogenicity, not only promote the understanding of its pathogenesis, but also provide important information for the prevention and control of dengue fever. It is of great significance in the pathogenesis of DENV, and for the development of vaccines and treatment drugs.
In the study of the pathogenesis of dengue virus and its antibody therapy strategy, it is found that cross reactive non neutralizing antibodies and subneutralizing antibodies can produce antibody dependent infection enhancement (ADE) through the Fc segment of the antibody, thereby promoting the progression of the disease. This phenomenon hinders the vaccine and monoclonal antibodies in dengue fever. And other related virus prevention and control applications. Dengue fever often occurs several serotypes of cross infection, therefore, the antibody must be used for treatment, must be able to produce effective protection of four serotype viruses. There are multiple cross reactive antibodies, but the protective titers for different serotype viruses are different, and the mixed application of several antibodies has complex components and many clinical trials need to be carried out. Dual-Variable-DomainImmunoglobulin (DVD-Ig) can be used to construct a variable region of 2 different antibodies in one. The antibody molecules of the plant maintain the affinity and protective activity of the original antibody, which greatly simplifies the trouble caused by the mixed application of multiple antibodies. The obtained antibodies can be further transformed by genetic engineering, and the Fc of the antibody is transformed to eliminate ADE. in the treatment of four serotype dengue viruses by targeting only a few types. The monoclonal antibodies with strong neutralization activity were rebuilt to obtain double variable region antibody against four serotype dengue viruses with broad spectrum and activity.
The aim of this study is to screen the neutralization activity of monoclonal antibodies with hybridoma screening technique, to verify the neutralization activity in vivo and in vitro and to further analyze the neutralization epitopes of the ED3 protein of dengue virus, and to clarify the key sites of antibody binding. A broad spectrum neutralizing antibody with no ADE activity is included. The study mainly consists of four parts.
Preparation and functional identification of specific monoclonal antibodies against dengue virus ED3 region
The hybridoma cell lines secreted against dengue virus (dengue virus) ED3 region were established with four serotypes of serotype dengue virus ED3 protein as immunogen, and 14 dengue virus ED3 specific monoclonal antibodies (1B12,1E12,1G6,1H8,2F9,2G9,2H12,3A10,3H12,4H10,5C10,6E1,6H7 and 7G5) were finally obtained.
In order to identify the virus targeted by these 14 monoclonal antibodies, we detected the immunofluorescence of the antigen prepared by the BHK cells infected by four serotype viruses. The results showed that the monoclonal antibodies secreted by these 14 hybridoma cells were specific against dengue virus and did not cross reaction to other members of the genus YhV; 14 monoclonal antibodies were against the virus. The cross reaction determination of different serotype dengue virus showed that 1B12,1E12,2H12,3A10,5C10,6H7 and 7G5 were specific monoclonal antibodies of dengue type 1 virus; 1H8 and 2G9 were dengue type 3 specific monoclonal antibodies; 1G6 was dengue type 4 virus specific monoclonal antibody; 2F9 could be crossed with dengue virus 1,3 virus; 3H12 and 4H10 can be cross combined with type 1-3 dengue virus; 6E1 intersecting These results suggest that we have obtained ED3 domain specific monoclonal antibodies against four serotype dengue viruses, which provide important information and tools for the antigen epitope analysis of ED3 protein, the diagnosis of dengue virus, and the research of new vaccines and antiviral drugs.
The neutralization activity of the 14 dengue virus specific monoclonal antibodies was detected by plaque neutralization and reduction test, and a specific neutralizing antibody of dengue type 4 virus was obtained. Secondly, the protective effect of the neutralization activity in vitro on the internal protection model of the milk rat was observed. The results showed that one of them was specific against dengue type 4 virus. The monoclonal antibody 1G6 has strong in vitro neutralization activity and in vivo protective effect. A single 50 g dose of antibody can survive dengue type 4 virus infection of 4H and 24h, respectively. It shows that it has a certain therapeutic effect on the infection of dengue virus.
In order to determine the mechanism of neutralizing antibody, we used the plaque assay before and after adsorption to determine its role in the virus infection cycle. The results showed that 1G6 played a neutralization effect mainly by inhibiting the adsorption of dengue virus and target cells, and when the virus was adsorbed with the target cells, the potency of the virus was greatly reduced.
Two, screening and identification of neutralization epitopes
In order to analyze the neutralization epitopes of dengue type 4 virus specific neutralizing antibodies, we first screened the epitopes of mAb 1G6 epitopes by using phage random 12 peptide library, and carried out a series of deletion mutations for every 5 amino acids to further confirm the screening results of the peptide library. Sequence alignment results showed that the monoclonal antibody table was a monoclonal antibody table. The corresponding polypeptide sequence is located in the 387LTLH390 region of the dengue type 4 virus E protein third domain. In the first order structure, the dengue type 4 virus E protein 388th and 390th amino acids are different from the dengue type 1-3 virus ED3 protein; from the three dimensional structure of the existing crystallization, it is found that the 390th bits of histidine, which is located in one of the other types, is found. The structure of the imidazole ring of the leather virus.
In order to determine the key site of the combination of monoclonal antibody 1G6 and E protein, we further carried out site directed mutagenesis and measured the effect of mutation of different amino acid sites on antibody binding power in indirect ELISA. The results showed that when T388G and H390G were mutated separately, the binding power of the monoclonal antibody 1G6 and the ED3 protein decreased significantly; when the two combined mutation, The complete loss of binding capacity indicates that the T388 and H390 sites of dengue virus E protein are the key sites for 1G6 binding.
Three, construction of chimeric antibody and its biological characteristics
In order to weaken the heterologous reaction of mouse antibody and construct a double variable region antibody with broad-spectrum and neutralization activity in the next step, the light heavy chain variable region sequence of mouse source antibody 1G6 was first obtained by 5 'RACE method, and the whole gene was synthesized with light and heavy chain variable region sequences with the antibody 1A1D-2 of the cross neutralization and dengue type 1-3 virus. The heavy chain variable regions were connected to the constant region of human heavy chain (IgG1) and light chain (kappa) to form a full-length human mouse chimeric antibody, respectively, to be loaded into the eukaryotic expression vector pcDNA3.1 (+) respectively. The expression vector of the heavy chain of chimeric antibody was constructed, and the CHO cells were transfected together. The stable secreting human mouse chimeric antibody was obtained by G418 pressure screening. The experiment proved that The antibody has the same antigen binding activity and in vitro neutralization activity as the parent mouse antibody, which lays the foundation for the construction of neutralizing antibodies in two variable regions.
Four, construction and functional identification of double variable region antibody without ADE activity.
Using gene engineering technology, the light and heavy chain variable region sequences of mAb 1A1D-2 and 1G6 were connected with 9 amino acid linker, respectively, and constructed into DVD1A1D-1G6-VL, DVD1G6-1A1D-VL and DVD1A1D-1G6-VH, DVD1G6-1A1D-VH fragments. They were connected to the light and heavy chain constant region of the human IgG 1 and loaded into the expression vector. The body pcDNA3.1 (+), CO transfected CHO cells, G418 screened to obtain 2 kinds of DVD antibody stable cell lines, no serum-free culture and antibody purification. In vitro binding experiment proved that the binding activity of DVD1A1D-1G6 was better than that of DVD1G6-1A1D. in vivo and in vitro. The DVD1A1D-1G6 antibody held the neutralization activity of the bivalent antibody in vivo and in vivo, and realized the resistance to disease. The activity of the four serotype dengue virus has been expanded, and the ADE activity of the Fc segment, which mediates the deletion of the ADE effect, disappears, and the mutation of the Fc segment has no obvious effect on the activity and activity, indicating that we successfully constructed a broad-spectrum anti dengue virus without ADE neutralization antibody.
In this study, we screened 14 dengue specific monoclonal antibodies, one of which was a new type of dengue type 4 virus specific neutralizing antibody, and found a new functional epitope of E protein. It laid the theoretical basis for elucidating the structure and function of E protein and the molecular mechanism of dengue virus disease and immunization; further construction obtained the same activity without ADE. The double variable region antibodies of four dengue viruses were neutralized and neutralized, which provided important information for developing anti dengue virus new antibody drugs.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 趙衛(wèi),范寶昌,胡志君,陳水平,王鵬程,苑錫同,李曉萸,于曼,秦鄂德,楊佩英;Study on the determinants of suckling mice neurovirulence of dengue 2 virus[J];Science in China(Series C:Life Sciences);2003年01期

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本文編號(hào)：1872056