【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，隨著旅游業(yè)的發(fā)展和地球溫暖化，世界范圍內DF和DHF/DSS的流行、暴發(fā)更加頻繁，影響范圍在不斷擴大，發(fā)病率不斷增加。據(jù)WHO最新統(tǒng)計，熱帶與亞熱帶地區(qū)每年有超過3.9億人感染DENV。值得一提的是，2013年夏季，我國廣東、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)布報告，認為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個開放讀碼框編碼三種結構蛋白包膜糖蛋白（E）、膜蛋白（M）和衣殼蛋白（C），以及七種非結構蛋白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構蛋白，在病毒吸附、與宿主細胞膜融合以及病毒組裝過程中具有重要的作用，是DENV吸附于靶細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包膜糖蛋白E與細胞膜以酸性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感染細胞內合成的NS1蛋白通過E蛋白C端末尾的疏水性信號序列進入內質網(wǎng)，隨后信號肽酶作用于E/NS1的連接處最終產生成熟的NS1蛋白。成熟的NS1蛋白以細胞內、細胞膜和細胞外分泌的形式存在，NS1蛋白有助于免疫復合物的形成，誘發(fā)機體產生保護性抗體[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，但對于血管通透性增加的機制尚未完全闡明。已知整合素家族是重要的細胞外基質蛋白，其中的整合素β3和β1主要分布在血管內皮細胞表面，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可以上調整合素β3的表達，且DENV E蛋白與整合素β3在HMEC-1內存在很明顯的共定位現(xiàn)象，利用RNAi技術下調整合素β3的表達可明顯抑制病毒的進入[8]，推測整合素β3可能是DENV入侵宿主細胞受體或共受體。但目前沒有直接證據(jù)證明E蛋白與整合素β3之間的相互作用。因此，本研究應用Octet系統(tǒng)對重組E蛋白與可能受體整合素β3的親和力進行分析，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疫苗誘導的免疫應答中起抑制作用[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系統(tǒng)表達E蛋白，純化DENV-2重組E蛋白后，對其免疫原性進行分析，同時設置GM-CSF佐劑組，研究GM-CSF在DENV E蛋白免疫中的作用。最后，我們利于實驗室已經(jīng)構建好的DENV prME、NS1+2a蛋白的真核表達質粒對動物的免疫保護作用進行了研究，希望能為DENV疫苗的研究奠定基礎。具體實驗結果如下： 一、DENV-2E蛋白的原核表達及免疫原性研究 1. DENV-2E蛋白的表達與純化 以pReceiver-E重組質粒(本室保存)為模板，擴增目的基因E胞外區(qū)基因片段，構建重組質粒E/pGEX-6P-1，轉化BL21菌，誘導表達，并摸索得到可溶性蛋白表達量相對較高的表達條件：誘導物IPTG濃度0.5mM，誘導溫度20℃，誘導時間為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法測定各組抗體效價；PRNT50檢測血清的抗DENV-2中和抗體效價；ELISpot法檢測脾細胞因子INF-γ、IL-2、IL-4、IL-10和IL-17水平。結果顯示：重組E蛋白組抗體效價為1:3,200，中和抗體效價為1;320；而E+pCAG-GM組抗體效價為1:6,400，中和抗體效價為1:1,280，說明E蛋白可以誘導產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Th1型細胞因子，IL-4和IL-10為Th2型細胞因子，IL-17為Th17細胞產生的細胞因子。細胞因子水平變化說明E蛋白組和E+pCAG-GM組誘導了Th1、Th2和Th17途徑的免疫應答反應。 3.重組E蛋白對小鼠的保護作用 同上方法免疫6周齡的BALB/c雌鼠，設置E蛋白組、E+pCAG-GM組、GST和生理鹽水組。三次免疫2周后，顱內注射1,000PFU的DENV-2，觀察小鼠死亡情況。結果為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%；結果說明，，重組E蛋白有一定的免疫保護作用，pCAG-GM有抑制E蛋白誘導的免疫應答的作用。 二、Octet系統(tǒng)分析DENV-2E蛋白與整合素β3的親和力 運用基于光纖生物傳感器的生物膜層光學干涉技術（BLI，BioLayerInterferometry）進行重組E蛋白與整合素β3的分子間的動力學分析，以確定重組E蛋白與整合素β3之間親和力的強弱和特異性。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)為易結合、難解離的特點，說明重組E蛋白與整合素β3有較強的特異性親和力。 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的方法測定各組抗體效價；PRNT50檢測血清的抗DENV-2中和抗體效價； ELISpot法檢測脾細胞因子INF-γ、IL-2、IL-4、IL-10、IL-17水平。結果：E蛋白組抗體效價是1:3,200，中和抗體效價為1:320；E+pRe-prM-E組為1:800，中和抗體效價為1:160；pRe-prM-E+pRe-NS1-NS2a組，抗體效價最高，可達到1:12,800，中和抗體效價也是最高為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。細胞因子水平說明pRe-prM-E和pRe-NS1-NS2a同時誘導了Th1和Th2途徑，且Th17途徑也參與了免疫應答過程；結果說明pRe-prM-E+pRe-NS1-NS2a組誘導的免疫應答水平明顯優(yōu)于其它組。 2. prME和NS1+2a DNA對小鼠保護作用的研究 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)合免疫質粒pRe-prM-E與pRe-NS1-NS2a組，整個實驗過程小鼠均未見任何發(fā)病癥狀，存活率為100%。結果說明，pRe-prM-E和pRe-NS1-NS2a質粒聯(lián)合免疫小鼠后，對小鼠的保護作用最好，可考慮作為候選DNA疫苗的分子靶點。 綜上，本實驗結果證明了E蛋白DENV在感染過程中的重要作用，動物實驗表明同時免疫prME和NS1+2a DNA可以能夠達到100%的免疫保護，可考慮作為候選疫苗的分子靶點。本研究為進一步研究DENV疫苗奠定了一定的基礎。
[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.
【學位授予單位】：首都醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.8

【共引文獻】

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本文編號：2179857