我国登革3型病毒2009年义乌流行株生物学特性研究
发布时间：2018-08-16 07:46
【摘要】：登革病毒(Dengue viruses, DENV)是一类严重危害人类健康的虫媒病毒,包括四个血清型(DENV1～4),该病毒主要通过伊蚊叮咬传播。据统计,全世界有超过25亿人处于登革病毒的威胁之中,登革病毒感染已经成为严重的世界性公共卫生问题。由于不同血清型登革病毒刺激机体产生的抗体不能起到交叉保护作用以及存在ADE效应,给登革疫苗的研制带来了极大挑战。目前登革病毒的预防主要依赖于切断蚊媒传播,尚无被批准的商业化疫苗。病毒样颗粒(Virus-like particles, VLPs)由于具有与天然病毒相似的免疫原性而无感染性,是一种较为理想的候选疫苗抗原。 登革病毒为单正链RNA病毒,基因组长约11kb,含单一开放读码框,依次编码3种结构蛋白(C、prM/M和E)和7种非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。E蛋白为主要的包膜糖蛋白,是登革病毒的主要保护性抗原。prM蛋白是膜蛋白M的前体,是形成病毒颗粒的重要膜蛋白并且有助于E蛋白结构的稳定。prM和E双基因的联合表达可以形成VLPs。 2009年7月,我国浙江省义乌市爆发了登革热疫情,经实验室诊断为登革3型病毒感染所致。本研究对2009年登革3型病毒义乌流行株进行系统进化分析,构建基于prM/E基因的登革毒样颗粒分泌表达质粒,在哺乳动物表达系统中获得了分泌表达的登革VLPs并对其免疫原性进行初步研究,为登革病毒样颗粒疫苗的研究及登革病毒诊断抗原的制备奠定了基础。 本研究主要分为三部分： 一、登革3型病毒2009年义乌流行株系统进化分析 根据浙江省义乌市登革病毒流行资料进行统计学分析,描述其流行病学特征。提取义乌流行株病毒RNA,利用RT-PCR法获得登革3型病毒义乌流行株全基因组序列。从GenBank中选取不同地区不同年份的60株登革3型病毒全基因组序列,并选择登革病毒中国流行株(其中3株登革1型,2株登革2型,1株登革4型)作为外群,用MEGA4进行序列比对及同源性分析,采用Neighbor-joining (NJ)法建立系统发生树,并用Bootstrap值为1000评价系统发生树。系统进化分析显示义乌流行株与广州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别为99.76%和99.64%。义乌流行株属于登革3型病毒亚型Ⅲ。 二、利用哺乳动物表达系统表达登革3型病毒VLPs PCR获得prM/E基因,通过融合PCR的方法在prM基因前添加乙型脑炎病毒的信号肽序列并同时删除E蛋白羧基末端的20%或替换为乙脑病毒E蛋白相应的部分,获得基因JprME、JprME397、JprME397J,分别克隆入真核表达载体pcDNA5/FRT中,经过酶切鉴定和测序鉴定后,获得三种读码框架及序列正确的重组质粒D3YWJprME-pcDNA5/FRT, D3YWJprME397-pcDNA5/FRT, D3YWJprME397J-pcDNA5/FRT。运用脂质体法分别将重组质粒转染293T细胞进行瞬时表达,利用间接免疫荧光法和Western印迹法分别检测prME蛋白在293T细胞中的表达及分泌情况。结果显示三种重组质粒转染293T细胞后均有登革3型病毒蛋白的表达。转染了D3YWJprME-pcDNA5/FRT重组质粒的293T细胞上清中没有特异的E蛋白条带,但经基因改造后的重组质粒D3YWJprME397-pcDNA5/FRT和D3YWJprME397J-pcDNA5/FRT的细胞上清中均可检测到登革3型病毒特异的E蛋白条带。故转染了三种重组质粒的293T细胞均可表达登革3型病毒prM/E蛋白,但登革病毒E蛋白羧基末端的20%影响其蛋白的分泌。在293T细胞中大量转染D3YWJprME397J-pcDNA5/FRT,将上清通过浓缩及蔗糖密度梯度离心获得纯化的D3YWVLPs。 三、D3YWVLPs免疫原性研究 将4～6周龄的BALB/c雌性小鼠随机分为5组(5只／组),即登革3型义乌流行株病毒样颗粒组(YW-VLPs),登革3型义乌流行株灭活病毒组(YW-V),登革3型国际标准株病毒样颗粒组(H87-VLPs),登革3型国际标准株灭活病毒组(H87-V),PBS对照组(PBS)。将抗原及PBS分别与等体积佐剂充分乳化,于第0,14,28天进行腹腔接种,免疫剂量为100μg/只。体液免疫及细胞免疫检测结果显示,VLPs能够刺激机体产生有效的免疫反应,但VLPs组与灭活病毒组免疫效果无统计学差异(p0.05),义乌流行株与国际标准株间无论在VLPs组还是灭活病毒组免疫效果均无统计学差异(p0.05)。义乌流行株VLPs可刺激小鼠产生针对DENV1～4EⅢ蛋白的IgG抗体,但仅具有中和登革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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
本文编号：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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
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相关期刊论文 前2条
1 吴德仁;谢平;;我国亚热带地区登革热流行概况[J];应用预防医学;2009年03期
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