我國登革3型病毒2009年義烏流行株生物學(xué)特性研究
發(fā)布時(shí)間：2018-08-16 07:46
【摘要】：登革病毒(Dengue viruses, DENV)是一類嚴(yán)重危害人類健康的蟲媒病毒,包括四個(gè)血清型(DENV1～4),該病毒主要通過伊蚊叮咬傳播。據(jù)統(tǒng)計(jì),全世界有超過25億人處于登革病毒的威脅之中,登革病毒感染已經(jīng)成為嚴(yán)重的世界性公共衛(wèi)生問題。由于不同血清型登革病毒刺激機(jī)體產(chǎn)生的抗體不能起到交叉保護(hù)作用以及存在ADE效應(yīng),給登革疫苗的研制帶來了極大挑戰(zhàn)。目前登革病毒的預(yù)防主要依賴于切斷蚊媒傳播,尚無被批準(zhǔn)的商業(yè)化疫苗。病毒樣顆粒(Virus-like particles, VLPs)由于具有與天然病毒相似的免疫原性而無感染性,是一種較為理想的候選疫苗抗原。 登革病毒為單正鏈RNA病毒,基因組長約11kb,含單一開放讀碼框,依次編碼3種結(jié)構(gòu)蛋白(C、prM/M和E)和7種非結(jié)構(gòu)蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。E蛋白為主要的包膜糖蛋白,是登革病毒的主要保護(hù)性抗原。prM蛋白是膜蛋白M的前體,是形成病毒顆粒的重要膜蛋白并且有助于E蛋白結(jié)構(gòu)的穩(wěn)定。prM和E雙基因的聯(lián)合表達(dá)可以形成VLPs。 2009年7月,我國浙江省義烏市爆發(fā)了登革熱疫情,經(jīng)實(shí)驗(yàn)室診斷為登革3型病毒感染所致。本研究對2009年登革3型病毒義烏流行株進(jìn)行系統(tǒng)進(jìn)化分析,構(gòu)建基于prM/E基因的登革毒樣顆粒分泌表達(dá)質(zhì)粒,在哺乳動物表達(dá)系統(tǒng)中獲得了分泌表達(dá)的登革VLPs并對其免疫原性進(jìn)行初步研究,為登革病毒樣顆粒疫苗的研究及登革病毒診斷抗原的制備奠定了基礎(chǔ)。 本研究主要分為三部分： 一、登革3型病毒2009年義烏流行株系統(tǒng)進(jìn)化分析 根據(jù)浙江省義烏市登革病毒流行資料進(jìn)行統(tǒng)計(jì)學(xué)分析,描述其流行病學(xué)特征。提取義烏流行株病毒RNA,利用RT-PCR法獲得登革3型病毒義烏流行株全基因組序列。從GenBank中選取不同地區(qū)不同年份的60株登革3型病毒全基因組序列,并選擇登革病毒中國流行株(其中3株登革1型,2株登革2型,1株登革4型)作為外群,用MEGA4進(jìn)行序列比對及同源性分析,采用Neighbor-joining (NJ)法建立系統(tǒng)發(fā)生樹,并用Bootstrap值為1000評價(jià)系統(tǒng)發(fā)生樹。系統(tǒng)進(jìn)化分析顯示義烏流行株與廣州GZ1D3株(GB：GU363549)及印度GWL-25株(GB：AY770511)相似度最高,氨基酸序列同源性分別為99.76%和99.64%。義烏流行株屬于登革3型病毒亞型Ⅲ。 二、利用哺乳動物表達(dá)系統(tǒng)表達(dá)登革3型病毒VLPs PCR獲得prM/E基因,通過融合PCR的方法在prM基因前添加乙型腦炎病毒的信號肽序列并同時(shí)刪除E蛋白羧基末端的20%或替換為乙腦病毒E蛋白相應(yīng)的部分,獲得基因JprME、JprME397、JprME397J,分別克隆入真核表達(dá)載體pcDNA5/FRT中,經(jīng)過酶切鑒定和測序鑒定后,獲得三種讀碼框架及序列正確的重組質(zhì)粒D3YWJprME-pcDNA5/FRT, D3YWJprME397-pcDNA5/FRT, D3YWJprME397J-pcDNA5/FRT。運(yùn)用脂質(zhì)體法分別將重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞進(jìn)行瞬時(shí)表達(dá),利用間接免疫熒光法和Western印跡法分別檢測prME蛋白在293T細(xì)胞中的表達(dá)及分泌情況。結(jié)果顯示三種重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞后均有登革3型病毒蛋白的表達(dá)。轉(zhuǎn)染了D3YWJprME-pcDNA5/FRT重組質(zhì)粒的293T細(xì)胞上清中沒有特異的E蛋白條帶,但經(jīng)基因改造后的重組質(zhì)粒D3YWJprME397-pcDNA5/FRT和D3YWJprME397J-pcDNA5/FRT的細(xì)胞上清中均可檢測到登革3型病毒特異的E蛋白條帶。故轉(zhuǎn)染了三種重組質(zhì)粒的293T細(xì)胞均可表達(dá)登革3型病毒prM/E蛋白,但登革病毒E蛋白羧基末端的20%影響其蛋白的分泌。在293T細(xì)胞中大量轉(zhuǎn)染D3YWJprME397J-pcDNA5/FRT,將上清通過濃縮及蔗糖密度梯度離心獲得純化的D3YWVLPs。 三、D3YWVLPs免疫原性研究 將4～6周齡的BALB/c雌性小鼠隨機(jī)分為5組(5只／組),即登革3型義烏流行株病毒樣顆粒組(YW-VLPs),登革3型義烏流行株滅活病毒組(YW-V),登革3型國際標(biāo)準(zhǔn)株病毒樣顆粒組(H87-VLPs),登革3型國際標(biāo)準(zhǔn)株滅活病毒組(H87-V),PBS對照組(PBS)。將抗原及PBS分別與等體積佐劑充分乳化,于第0,14,28天進(jìn)行腹腔接種,免疫劑量為100μg/只。體液免疫及細(xì)胞免疫檢測結(jié)果顯示,VLPs能夠刺激機(jī)體產(chǎn)生有效的免疫反應(yīng),但VLPs組與滅活病毒組免疫效果無統(tǒng)計(jì)學(xué)差異(p0.05),義烏流行株與國際標(biāo)準(zhǔn)株間無論在VLPs組還是滅活病毒組免疫效果均無統(tǒng)計(jì)學(xué)差異(p0.05)。義烏流行株VLPs可刺激小鼠產(chǎn)生針對DENV1～4EⅢ蛋白的IgG抗體,但僅具有中和登革3型病毒的活性。
[Abstract]:Dengue viruses (DENV) are a group of arboviruses that seriously endanger human health, including four serotypes (DENV1-4). The virus is mainly transmitted by Aedes mosquito bites. According to statistics, more than 2.5 billion people worldwide are under the threat of dengue virus. Dengue virus infection has become a serious public health problem worldwide. Antibodies produced by different serotypes of dengue virus can not cross-protect the body and have the ADE effect, which poses a great challenge to the development of dengue vaccines. At present, the prevention of dengue virus mainly depends on cutting off mosquito vector transmission, and there is no commercial vaccine approved yet. Virus-like particles (VLPs) are due to their presence. It has immunogenicity similar to natural virus without infection. It is an ideal candidate vaccine antigen.
Dengue virus is a single single open reading frame RNA virus with a genome length of about 11 kb. It encodes three structural proteins (C, prM/M and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in turn. E protein is the main envelope glycoprotein and the main protective antigen of dengue virus. The combination of prM and E genes can form VLPs.
In July 2009, an outbreak of dengue fever occurred in Yiwu City, Zhejiang Province, China. The dengue virus type 3 strain was diagnosed to be infected by Dengue virus type 3 in laboratory. In this study, the epidemic strain of Dengue virus type 3 in Yiwu in 2009 was systematically analyzed, and the secretory expression plasmid of dengue virus-like particles based on prM/E gene was constructed. Dengue VLPs and their immunogenicity were preliminarily studied, which laid a foundation for the study of dengue virus-like granular vaccine and preparation of dengue virus diagnostic antigen.
This study is divided into three parts.
Phylogenetic analysis of dengue 3 virus in Yiwu in 2009
According to the epidemic data of dengue virus in Yiwu City of Zhejiang Province, the epidemiological characteristics were described. RNA of the epidemic strain of Dengue virus in Yiwu City was extracted and the whole genome sequence of the epidemic strain of Dengue virus type 3 was obtained by RT-PCR. The genome sequences of 60 dengue virus strains from different regions and different years were selected from GenBank. The phylogenetic tree was established by Neighbor-joining (NJ) method and the phylogenetic tree was evaluated by Bootstrap value of 1000. Phylogenetic analysis showed that the epidemic strains of Yiwu and Guangzhou GZ1D3 (GB: GU36) were identified by phylogenetic analysis. 3549 and GWL-25 strains from India (GB:AY770511) had the highest similarity, with 99.76% and 99.64% amino acid sequence homology, respectively.
Two, expression of dengue 3 virus VLPs by mammalian expression system.
The prM/E gene was obtained by PCR. The prM/E gene was fused with the signal peptide sequence of Japanese encephalitis virus (JEV) before the prM gene and 20% of the carboxyl end of the E protein was deleted or replaced with the corresponding part of the E protein of Japanese encephalitis virus (JEV). The genes JprME, JprME397 and JprME397J were cloned into the eukaryotic expression vector pcDNA5/FRT and identified by enzyme digestion. The recombinant plasmids D3YWJprME-pcDNA5/FRT, D3YWJprME397-pcDNA5/FRT and D3YWJprME397J-pcDNA5/FRT with correct reading frame and sequence were obtained. The recombinant plasmids were transfected into 293T cells by liposome method for transient expression. The prME protein in 293T cells was detected by indirect immunofluorescence and Western blotting respectively. The results showed that all the three recombinant plasmids expressed dengue-3 virus protein in 293T cells. No specific E-protein band was found in the supernatant of 293T cells transfected with D3YWJprME-pcDNA5/FRT recombinant plasmid, but the recombinant plasmids D3YWJprME397-pcDNA5/FRT and D3YWJprME397J-pcDNA5/FRT were genetically modified. The specific E protein bands of dengue virus type 3 were detected in the supernatant. Therefore, 293T cells transfected with three recombinant plasmids could express the prM/E protein of dengue virus type 3, but 20% of the carboxyl terminal of the E protein of dengue virus affected the secretion of the protein. Purified D3YWVLPs. was obtained by gradient centrifugation.
Three, D3YWVLPs immunogenicity study
Female BALB/c mice aged 4-6 weeks were randomly divided into 5 groups (5 mice/group), namely, YW-VLPs, YW-V, H87-VLPs, H87-VLPs, H87-V, PBS control group. PBS was fully emulsified with the same volume adjuvant and inoculated intraperitoneally at 0, 14 and 28 days. The immune dosage was 100 UG / mouse. The results of humoral and cellular immunity tests showed that VLPs could stimulate the body to produce effective immune response, but there was no significant difference in the immune effect between the VLPs group and the inactivated virus group (p0.05). There was no significant difference in the immune effect between the VLPs group and the inactivated virus group (p0.05). VLPs of Yiwu epidemic strain could stimulate mice to produce IgG antibodies against DENV1-4E III protein, but only had the activity of neutralizing dengue virus type 3.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R373
本文編號：2185351
[Abstract]:Dengue viruses (DENV) are a group of arboviruses that seriously endanger human health, including four serotypes (DENV1-4). The virus is mainly transmitted by Aedes mosquito bites. According to statistics, more than 2.5 billion people worldwide are under the threat of dengue virus. Dengue virus infection has become a serious public health problem worldwide. Antibodies produced by different serotypes of dengue virus can not cross-protect the body and have the ADE effect, which poses a great challenge to the development of dengue vaccines. At present, the prevention of dengue virus mainly depends on cutting off mosquito vector transmission, and there is no commercial vaccine approved yet. Virus-like particles (VLPs) are due to their presence. It has immunogenicity similar to natural virus without infection. It is an ideal candidate vaccine antigen.
Dengue virus is a single single open reading frame RNA virus with a genome length of about 11 kb. It encodes three structural proteins (C, prM/M and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in turn. E protein is the main envelope glycoprotein and the main protective antigen of dengue virus. The combination of prM and E genes can form VLPs.
In July 2009, an outbreak of dengue fever occurred in Yiwu City, Zhejiang Province, China. The dengue virus type 3 strain was diagnosed to be infected by Dengue virus type 3 in laboratory. In this study, the epidemic strain of Dengue virus type 3 in Yiwu in 2009 was systematically analyzed, and the secretory expression plasmid of dengue virus-like particles based on prM/E gene was constructed. Dengue VLPs and their immunogenicity were preliminarily studied, which laid a foundation for the study of dengue virus-like granular vaccine and preparation of dengue virus diagnostic antigen.
This study is divided into three parts.
Phylogenetic analysis of dengue 3 virus in Yiwu in 2009
According to the epidemic data of dengue virus in Yiwu City of Zhejiang Province, the epidemiological characteristics were described. RNA of the epidemic strain of Dengue virus in Yiwu City was extracted and the whole genome sequence of the epidemic strain of Dengue virus type 3 was obtained by RT-PCR. The genome sequences of 60 dengue virus strains from different regions and different years were selected from GenBank. The phylogenetic tree was established by Neighbor-joining (NJ) method and the phylogenetic tree was evaluated by Bootstrap value of 1000. Phylogenetic analysis showed that the epidemic strains of Yiwu and Guangzhou GZ1D3 (GB: GU36) were identified by phylogenetic analysis. 3549 and GWL-25 strains from India (GB:AY770511) had the highest similarity, with 99.76% and 99.64% amino acid sequence homology, respectively.
Two, expression of dengue 3 virus VLPs by mammalian expression system.
The prM/E gene was obtained by PCR. The prM/E gene was fused with the signal peptide sequence of Japanese encephalitis virus (JEV) before the prM gene and 20% of the carboxyl end of the E protein was deleted or replaced with the corresponding part of the E protein of Japanese encephalitis virus (JEV). The genes JprME, JprME397 and JprME397J were cloned into the eukaryotic expression vector pcDNA5/FRT and identified by enzyme digestion. The recombinant plasmids D3YWJprME-pcDNA5/FRT, D3YWJprME397-pcDNA5/FRT and D3YWJprME397J-pcDNA5/FRT with correct reading frame and sequence were obtained. The recombinant plasmids were transfected into 293T cells by liposome method for transient expression. The prME protein in 293T cells was detected by indirect immunofluorescence and Western blotting respectively. The results showed that all the three recombinant plasmids expressed dengue-3 virus protein in 293T cells. No specific E-protein band was found in the supernatant of 293T cells transfected with D3YWJprME-pcDNA5/FRT recombinant plasmid, but the recombinant plasmids D3YWJprME397-pcDNA5/FRT and D3YWJprME397J-pcDNA5/FRT were genetically modified. The specific E protein bands of dengue virus type 3 were detected in the supernatant. Therefore, 293T cells transfected with three recombinant plasmids could express the prM/E protein of dengue virus type 3, but 20% of the carboxyl terminal of the E protein of dengue virus affected the secretion of the protein. Purified D3YWVLPs. was obtained by gradient centrifugation.
Three, D3YWVLPs immunogenicity study
Female BALB/c mice aged 4-6 weeks were randomly divided into 5 groups (5 mice/group), namely, YW-VLPs, YW-V, H87-VLPs, H87-VLPs, H87-V, PBS control group. PBS was fully emulsified with the same volume adjuvant and inoculated intraperitoneally at 0, 14 and 28 days. The immune dosage was 100 UG / mouse. The results of humoral and cellular immunity tests showed that VLPs could stimulate the body to produce effective immune response, but there was no significant difference in the immune effect between the VLPs group and the inactivated virus group (p0.05). There was no significant difference in the immune effect between the VLPs group and the inactivated virus group (p0.05). VLPs of Yiwu epidemic strain could stimulate mice to produce IgG antibodies against DENV1-4E III protein, but only had the activity of neutralizing dengue virus type 3.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 吳德仁;謝平;;我國亞熱帶地區(qū)登革熱流行概況[J];應(yīng)用預(yù)防醫(yī)學(xué);2009年03期
2 蒙中秋;全球登革熱/登革出血熱的流行態(tài)勢及我國口岸監(jiān)測管理[J];中國熱帶醫(yī)學(xué);2005年07期
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