【摘要】：登革病毒（Dengue virus，DENV）是登革熱(classical dengue fever，DF)和登革出血熱/登革休克綜合癥(dengue hemorrhagic fever/dengue shock syndrome，DHF/DSS)的病原體[1]。近年來(lái)，隨著旅游業(yè)的發(fā)展和地球溫暖化，世界范圍內(nèi)DF和DHF/DSS的流行、暴發(fā)更加頻繁，影響范圍在不斷擴(kuò)大，發(fā)病率不斷增加。據(jù)WHO最新統(tǒng)計(jì)，熱帶與亞熱帶地區(qū)每年有超過(guò)3.9億人感染DENV。值得一提的是，2013年夏季，我國(guó)廣東、云南有大范圍的DF疫情，且有較多的重癥病例。因而WHO再次發(fā)布報(bào)告，認(rèn)為DF已成為全球蔓延最快的病種，亟待研制安全有效的疫苗、特效抗病毒藥物進(jìn)行防治。 DENV屬于黃病毒科、黃病毒屬的單股正鏈RNA病毒，有四種血清型(DENV1-4）[2]，DENV基因組大約為11kb，1個(gè)開(kāi)放讀碼框編碼三種結(jié)構(gòu)蛋白包膜糖蛋白（E）、膜蛋白（M）和衣殼蛋白（C），以及七種非結(jié)構(gòu)蛋白NS1、NS2a、NS2b、NS3、NS4a、NS4b和NS5[3，4]。病毒表面的包膜蛋白E蛋白是DENV病毒體上的包膜糖蛋白和最大的結(jié)構(gòu)蛋白，在病毒吸附、與宿主細(xì)胞膜融合以及病毒組裝過(guò)程中具有重要的作用，是DENV吸附于靶細(xì)胞與受體相互作用的病毒表面特異性吸附蛋白[5]。prM蛋白是未成熟病毒顆粒中的膜蛋白前體，prM蛋白的N端序列被蛋白酶切除后即生成M蛋白，只有含M蛋白的成熟病毒顆粒才能介導(dǎo)包膜糖蛋白E與細(xì)胞膜以酸性pH依賴型的方式發(fā)生融合[6]。DENV NS1蛋白的分子量約為42~50KDa，在DENV感染細(xì)胞內(nèi)合成的NS1蛋白通過(guò)E蛋白C端末尾的疏水性信號(hào)序列進(jìn)入內(nèi)質(zhì)網(wǎng)，隨后信號(hào)肽酶作用于E/NS1的連接處最終產(chǎn)生成熟的NS1蛋白。成熟的NS1蛋白以細(xì)胞內(nèi)、細(xì)胞膜和細(xì)胞外分泌的形式存在，NS1蛋白有助于免疫復(fù)合物的形成，誘發(fā)機(jī)體產(chǎn)生保護(hù)性抗體[7]。 DHF/DSS是DENV感染的重癥，其特征性的臨床表現(xiàn)之一是血管通透性增加引起的滲出和出血，但對(duì)于血管通透性增加的機(jī)制尚未完全闡明。已知整合素家族是重要的細(xì)胞外基質(zhì)蛋白，其中的整合素β3和β1主要分布在血管內(nèi)皮細(xì)胞表面，在維持血管壁完整性上發(fā)揮著重要作用。課題組前期研究發(fā)現(xiàn)DENV-2感染HMEC-1可以上調(diào)整合素β3的表達(dá)，且DENV E蛋白與整合素β3在HMEC-1內(nèi)存在很明顯的共定位現(xiàn)象，利用RNAi技術(shù)下調(diào)整合素β3的表達(dá)可明顯抑制病毒的進(jìn)入[8]，推測(cè)整合素β3可能是DENV入侵宿主細(xì)胞受體或共受體。但目前沒(méi)有直接證據(jù)證明E蛋白與整合素β3之間的相互作用。因此，本研究應(yīng)用Octet系統(tǒng)對(duì)重組E蛋白與可能受體整合素β3的親和力進(jìn)行分析，初步研究了E蛋白與整合素β3之間的相互作用。 GM-CSF在JEV prME DNA疫苗誘導(dǎo)的免疫應(yīng)答中起抑制作用[9]，但其在DENV蛋白免疫中的作用尚不清楚。為此，本研究的第二部分工作是原核表達(dá)系統(tǒng)表達(dá)E蛋白，純化DENV-2重組E蛋白后，對(duì)其免疫原性進(jìn)行分析，同時(shí)設(shè)置GM-CSF佐劑組，研究GM-CSF在DENV E蛋白免疫中的作用。最后，我們利于實(shí)驗(yàn)室已經(jīng)構(gòu)建好的DENV prME、NS1+2a蛋白的真核表達(dá)質(zhì)粒對(duì)動(dòng)物的免疫保護(hù)作用進(jìn)行了研究，希望能為DENV疫苗的研究奠定基礎(chǔ)。具體實(shí)驗(yàn)結(jié)果如下： 一、DENV-2E蛋白的原核表達(dá)及免疫原性研究 1. DENV-2E蛋白的表達(dá)與純化 以pReceiver-E重組質(zhì)粒(本室保存)為模板，擴(kuò)增目的基因E胞外區(qū)基因片段，構(gòu)建重組質(zhì)粒E/pGEX-6P-1，轉(zhuǎn)化BL21菌，誘導(dǎo)表達(dá)，并摸索得到可溶性蛋白表達(dá)量相對(duì)較高的表達(dá)條件：誘導(dǎo)物IPTG濃度0.5mM，誘導(dǎo)溫度20℃，誘導(dǎo)時(shí)間為15h；親和層析純化重組E蛋白，最后得到純度較高的蛋白。 2.重組E蛋白免疫原性研究 免疫6周齡的BALB/c雌鼠，設(shè)置E蛋白組、E+pCAG-GM組、GST和生理鹽水組。三次免疫2周后，斷尾取血，ELISA法測(cè)定各組抗體效價(jià)；PRNT50檢測(cè)血清的抗DENV-2中和抗體效價(jià)；ELISpot法檢測(cè)脾細(xì)胞因子INF-γ、IL-2、IL-4、IL-10和IL-17水平。結(jié)果顯示：重組E蛋白組抗體效價(jià)為1:3,200，中和抗體效價(jià)為1;320；而E+pCAG-GM組抗體效價(jià)為1:6,400，中和抗體效價(jià)為1:1,280，說(shuō)明E蛋白可以誘導(dǎo)產(chǎn)生特異性抗體和中和抗體；E，E+pCAG-GM組IFN-γ、IL-2、IL-10和IL-17水平明顯上升，與生理鹽水組比較差異顯著（p0.05）。由于IL-2和IFN-γ為T(mén)h1型細(xì)胞因子，IL-4和IL-10為T(mén)h2型細(xì)胞因子，IL-17為T(mén)h17細(xì)胞產(chǎn)生的細(xì)胞因子。細(xì)胞因子水平變化說(shuō)明E蛋白組和E+pCAG-GM組誘導(dǎo)了Th1、Th2和Th17途徑的免疫應(yīng)答反應(yīng)。 3.重組E蛋白對(duì)小鼠的保護(hù)作用 同上方法免疫6周齡的BALB/c雌鼠，設(shè)置E蛋白組、E+pCAG-GM組、GST和生理鹽水組。三次免疫2周后，顱內(nèi)注射1,000PFU的DENV-2，觀察小鼠死亡情況。結(jié)果為E+pCAG-GM組、GST組和生理鹽水組小鼠全部死亡，E蛋白組存活率為33.33%；結(jié)果說(shuō)明，，重組E蛋白有一定的免疫保護(hù)作用，pCAG-GM有抑制E蛋白誘導(dǎo)的免疫應(yīng)答的作用。 二、Octet系統(tǒng)分析DENV-2E蛋白與整合素β3的親和力 運(yùn)用基于光纖生物傳感器的生物膜層光學(xué)干涉技術(shù)（BLI，BioLayerInterferometry）進(jìn)行重組E蛋白與整合素β3的分子間的動(dòng)力學(xué)分析，以確定重組E蛋白與整合素β3之間親和力的強(qiáng)弱和特異性。Octet系統(tǒng)分析顯示重組E蛋白與整合素β3平衡解離常數(shù)(KD)為3.14×10-9M，解離速率常數(shù)(Kdis)為5.11×10-5s-1。提示重組E蛋白與整合素β3的相互作用表現(xiàn)為易結(jié)合、難解離的特點(diǎn)，說(shuō)明重組E蛋白與整合素β3有較強(qiáng)的特異性親和力。 三、 prME及NS1+2a DNA的小鼠免疫原性研究 1. prME和NS1+2a DNA的免疫原性研究 6周齡BALB/c雌鼠分別免疫E蛋白、 E+pRe-prM-E、pRe-prM-E+pRe-NS1-NS2a，生理鹽水組注射等量生理鹽水。小鼠三次免疫2周后， ELISA的方法測(cè)定各組抗體效價(jià)；PRNT50檢測(cè)血清的抗DENV-2中和抗體效價(jià)； ELISpot法檢測(cè)脾細(xì)胞因子INF-γ、IL-2、IL-4、IL-10、IL-17水平。結(jié)果：E蛋白組抗體效價(jià)是1:3,200，中和抗體效價(jià)為1:320；E+pRe-prM-E組為1:800，中和抗體效價(jià)為1:160；pRe-prM-E+pRe-NS1-NS2a組，抗體效價(jià)最高，可達(dá)到1:12,800，中和抗體效價(jià)也是最高為1:2,560，并且pRe-prM-E+pRe-NS1-NS2a組INF-γ、IL-10和IL-17因子水平明顯高于其他組（p0.05）；IL-4因子水平，各組間差異不顯著。細(xì)胞因子水平說(shuō)明pRe-prM-E和pRe-NS1-NS2a同時(shí)誘導(dǎo)了Th1和Th2途徑，且Th17途徑也參與了免疫應(yīng)答過(guò)程；結(jié)果說(shuō)明pRe-prM-E+pRe-NS1-NS2a組誘導(dǎo)的免疫應(yīng)答水平明顯優(yōu)于其它組。 2. prME和NS1+2a DNA對(duì)小鼠保護(hù)作用的研究 將6周齡BALB/c雌鼠分別免疫E蛋白、 E+pRe-prM-E、pRe-prM-E+pRe-NS1-NS2a，生理鹽水組注射等量生理鹽水。三次免疫2周后，顱內(nèi)注射1,000PFU的DENV-2，觀察小鼠死亡情況。攻毒后第13d后，生理鹽水組小鼠全部死亡；E蛋白組和E+pRe-prM-E組均在第12d出現(xiàn)死亡，最終存活率均為33.3%；而聯(lián)合免疫質(zhì)粒pRe-prM-E與pRe-NS1-NS2a組，整個(gè)實(shí)驗(yàn)過(guò)程小鼠均未見(jiàn)任何發(fā)病癥狀，存活率為100%。結(jié)果說(shuō)明，pRe-prM-E和pRe-NS1-NS2a質(zhì)粒聯(lián)合免疫小鼠后，對(duì)小鼠的保護(hù)作用最好，可考慮作為候選DNA疫苗的分子靶點(diǎn)。 綜上，本實(shí)驗(yàn)結(jié)果證明了E蛋白DENV在感染過(guò)程中的重要作用，動(dòng)物實(shí)驗(yàn)表明同時(shí)免疫prME和NS1+2a DNA可以能夠達(dá)到100%的免疫保護(hù)，可考慮作為候選疫苗的分子靶點(diǎn)。本研究為進(jìn)一步研究DENV疫苗奠定了一定的基礎(chǔ)。
[Abstract]:Dengue virus (DENV) is the pathogen of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)[1].In recent years, with the development of tourism and the warming of the earth, the prevalence of DF and DHF/DSS worldwide has become more frequent and influential. According to the latest statistics of WHO, more than 390 million people are infected with DENV every year in tropical and subtropical regions. It is worth mentioning that in the summer of 2013, Guangdong and Yunnan in China had a wide range of DF epidemics and a large number of severe cases. Therefore, the WHO issued a report again that DF has become the fastest spreading disease in the world. It is urgent to develop a safe and effective vaccine for the prevention and treatment of specific antiviral drugs.
DENV belongs to the family Flaviviridae. The flavivirus is a single stranded positive stranded RNA virus. It has four serotypes (DENV1-4) [2], a DENV genome of about 11 kb, an open reading frame encoding three structural protein envelope glycoproteins (E), membrane protein (M) and capsid protein (C), and seven non-structural proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 [3, 4]. Protein E is the envelope glycoprotein and the largest structural protein of DENV. It plays an important role in virus adsorption, fusion with host cell membrane and virus assembly. It is a virus surface-specific adsorbent protein that DENV adsorbs on target cells interacting with receptors [5]. The N-terminal sequence of prM protein was excised by proteinase to produce M protein. Only mature virus particles containing M protein could mediate the fusion of envelope glycoprotein E with cell membrane in an acidic pH-dependent manner [6]. The water-borne signal sequence enters the endoplasmic reticulum, and then the signal peptidase acts on the junction of E/NS1 to produce mature NS1 protein. The mature NS1 protein exists in the form of intracellular, cell membrane and extracellular secretion. NS1 protein contributes to the formation of immune complex and induces the body to produce protective antibodies [7].
DHF/DSS is a severe infection of DENV. One of the clinical manifestations of DHF/DSS is exudation and hemorrhage caused by increased vascular permeability. However, the mechanism of increased vascular permeability has not been fully elucidated. Previous studies have shown that DENV-2 infection with HMEC-1 can up-regulate the expression of integrin beta-3, and the co-localization of DENV-E protein and integrin beta-3 is obvious in HMEC-1. Adjusting the expression of integrin beta-3 by RNAi technology can significantly inhibit the entry of the virus [8], suggesting that integrin beta-3 may be able. However, there is no direct evidence to prove the interaction between E protein and integrin beta 3. Therefore, the affinity between recombinant E protein and integrin beta 3 was analyzed by Octet system, and the interaction between E protein and integrin beta 3 was preliminarily studied.
GM-CSF can inhibit the immune response induced by JEV prME DNA vaccine [9], but its role in DENV protein immunity is not clear. Therefore, the second part of this study is to express E protein in prokaryotic expression system, purify recombinant E protein of DENV-2, analyze its immunogenicity, and set up GM-CSF adjuvant group to study the role of GM-CSF in DENV protein immunity. Finally, we will benefit from the construction of DENV prME, NS1 + 2A protein eukaryotic expression plasmid to study the immune protection of animals, hoping to lay the foundation for the study of DENV vaccine.
Prokaryotic expression and immunogenicity of DENV-2E protein
1. expression and purification of DENV-2E protein
The recombinant plasmid E/pGEX-6P-1 was constructed and transformed into BL21 strain to induce the expression of the soluble protein. The expression conditions were as follows: the concentration of IPTG was 0.5mM, the induction temperature was 20 C, the induction time was 15 h; the affinity chromatography was used to purify the recombinant plasmid E/pGEX-6P-1. The recombinant E protein was obtained, and finally the protein with high purity was obtained.
2. immunogenicity of recombinant E protein
Immunized 6-week-old BALB/c female rats were divided into E protein group, E+pCAG-GM group, GST group and normal saline group. After 2 weeks of immunization, blood samples were taken from the tail and the antibody titers were determined by ELISA; PRNT50 was used to detect the antibody titers against DENV-2; ELISpot was used to detect the levels of spleen cytokines INF-gamma, IL-2, IL-4, IL-10 and IL-17. The titer of antibody was 1:3,200, and the titer of neutralizing antibody was 1:320; the titer of antibody was 1:6,400 in E+pCAG-GM group and 1:1,280 in E+pCAG-GM group, indicating that E protein could induce specific antibody and neutralizing antibody; the levels of IFN-gamma, IL-2, IL-10 and IL-17 in E, E+pCAG-GM group were significantly higher than those in normal saline group (p0.05). FN-gamma is a Th1 type cytokine, IL-4 and IL-10 are Th2 type cytokines, and IL-17 is a Th17 type cytokine. The changes of cytokine levels indicate that E protein group and E+pCAG-GM group induce the immune responses of Th1, Th2 and Th17 pathways.
3. protective effect of recombinant E protein on mice
After 2 weeks of immunization, 1,000 PFU DENV-2 was injected into the brain to observe the death of BALB/c mice. The results showed that all the mice in E+pCAG-GM group, GST group and normal saline group died and the survival rate of E protein group was 33.33%. PCAG-GM can inhibit the immune response induced by E protein.
Two, the affinity of DENV-2E protein to integrin beta 3 was analyzed by Octet system.
Bio-Layer Interferometry (BLI) based on fiber optic biosensor was used to analyze the molecular dynamics of recombinant E protein and integrin beta 3 in order to determine the affinity and specificity between recombinant E protein and integrin beta 3. Octet system analysis showed that the equilibrium dissociation of recombinant E protein and integrin beta 3 was normal. The number (KD) of recombinant E protein was 3.14 X 10-9M and the dissociation rate constant (Kdis) was 5.11 X 10-5s-1. It was suggested that the interaction between recombinant E protein and integrin beta 3 was easy to combine and difficult to dissociate, indicating that recombinant E protein had a strong specific affinity with integrin beta 3.
Immunogenicity of three, prME and NS1+2a DNA mice
Immunogenicity of 1. prME and NS1+2a DNA
Six-week-old BALB/c female mice were immunized with E protein, E+pRe-prM-E, pRe-prM-E+pRe-NS1-NS2a, and saline group was injected with the same amount of saline. Two weeks after the mice were immunized three times, the antibody titers of each group were determined by ELISA; PRNT50 was used to detect the antibody titers against DENV-2 in serum; and the spleen cytokines INF-gamma, IL-2, IL-4, IL-10, IL-17 water were detected by ELISpot. Results: The titer of antibody in E protein group was 1:3,200, the titer of neutralizing antibody was 1:320, the titer of neutralizing antibody in E + pRe-prM-E group was 1:800, the titer of neutralizing antibody was 1:160, the titer of antibody in pRe-prM-E + pRe-NS1-NS2a group was 1:12,800, and the titer of neutralizing antibody was 1:2,560, and the levels of INF-gamma, IL-10 and IL-17 in pRe-prM-E + pRe-NS1-NS2a group were clear. The levels of cytokines showed that pRe-prM-E and pRe-NS1-NS2a induced both Th1 and Th2 pathways, and Th17 pathway also participated in the immune response process; the results showed that pRe-prM-E+pRe-NS1-NS2a induced immune response level was significantly higher than other groups.
Protective effects of 2. prME and NS1+2a DNA on mice
The 6-week-old BALB/c female rats were immunized with E protein, E+pRe-prM-E, pRe-prM-E+pRe-NS1-NS2a, and the normal saline group was injected with the same amount of saline. Two weeks after the third immunization, 1,000 PFU DENV-2 was injected into the brain to observe the death of the mice. The survival rate was 100%. The results showed that the combined immunization of pRe-prM-E and pRe-NS1-NS2a plasmids had the best protective effect on mice and could be considered as the molecular target of candidate DNA vaccine.
In conclusion, the results of this study demonstrated that E protein DENV played an important role in the process of infection. Animal experiments showed that prME and NS1+2a DNA could achieve 100% immune protection and could be considered as molecular targets of candidate vaccines.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R512.8

【共引文獻(xiàn)】

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