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1.
参照GenBank中的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14株序列设计了一对特异性引物,用PCR方法从SA14-14扩增E基因全长,然后克隆到pMD18-T载体中,转化宿主菌DH5a,提取阳性克隆质粒进行双酶切鉴定,将目的片段定向克隆到pET32a( )中,转化入BL21(DE3),经IPTG诱导可表达分子量约73ka的蛋白,Western blotting试验呈阳性,表明E基因得到表达。以纯化的表达产物为核心抗原,猪抗JEV血清为一抗,HRP标记羊抗猪IgG抗体为二抗建立间接ELISA方法,并初步检测了一些血清样品,结果提示表达的蛋白具有很好的应用开发价值。  相似文献   

2.
根据JEV病毒减毒株SA14—14—2基因组序列,设计覆盖全长的4对重叠引物,以提取的活疫苗病毒RNA为模板,RT—PCR扩增出4个片段,并克隆到质粒载体中,进一步构建两个半端分子克隆,然后将全长cDNA序列克隆到一个新改造的低拷贝质粒载体pBR—kpn中,构建我国流行性乙型脑炎病毒(JEV)基因组全长cDNA克隆。经过体外转录后得到的转录子转染BHK-21细胞,重新获得JEV的恢复病毒,通过生物学特性、分子生物学水平、蛋白水平等几个方面对恢复病毒进行鉴定。结果获得了稳定的全长cDNA克隆,转录子转染BHK-21细胞后,第4天开始出现细胞病变(CPE),第6~7天时CPE为 ,经过Vero细胞进一步放大培养后,间接免疫荧光实验和RT—PCR实验均为阳性。证实了构建的JEV的全长cDNA克隆有感染性,为进一步的研究奠定了基础。  相似文献   

3.
乙脑病毒持续感染株preM区序列分析   总被引:1,自引:0,他引:1  
为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒.利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较.preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸).对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同.认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关.  相似文献   

4.
为了研究乙型脑炎病毒持续感染株preM区域基因序列变异及其意义,我们将两种乙脑病毒野生株(JaGAr-01株和Nakayama株)分别感染人肝癌KN73细胞,经过多次细胞传代后建立乙脑病毒持续感染模型,收集感染细胞经反复冻融获取变异病毒。利用preM区特异引物进行RT-PCR法得到两种病毒的preM区基因片段,应用基因测序反应进行序列分析,并对两种病毒株preM区序列进行比较。preM区基因测序结果显示,与JaGAr-01野生株比较,JaGAr-01持续感染变异株(JaG-per)有1个核苷酸上碱基发生变异(第26位U→G)并导致相应氨基酸发生置换(第9位亮氨酸→精氨酸);Nakayama持续感染变异株(Nak-per)与其野生株相比则有11个核苷酸上碱基存在差异(第26位U→G,第37位G→A,第39位C→U,第45位U→C,第51位U→C,第99位U→C,第126位U→C,第165位C→U,第189位C→U,第195位C→U,第198位U→C),但仅有其中第26位、第37位、第39位的碱基变异引起相应编码的氨基酸发生置换(第9位亮氨酸→精氨酸及第13位缬氨酸→异亮氨酸)。对比还发现变异后的JaGAr-01持续感染株与Nakayama持续感染株的基因序列相同。认为乙脑病毒持续感染变异株preM区存在基因变异,这种变异可能与该区参与病毒持续感染及维持病毒生物学特性有关。  相似文献   

5.
流行性乙型脑炎是由乙型脑炎病毒引起的、经蚊虫传播的严重危害中枢神经系统的人畜共患急性传染病,其重症病死率高,易造成永久性的神经系统后遗症,严重威胁着人类的健康。目前尚无特效的治疗流行性乙型脑炎的方法,控制蚊虫传播和免疫接种是当前的主要防御手段。简要综述了乙型脑炎病毒的基因组结构、结构蛋白与非结构蛋白功能、基因分型,以及流行性乙型脑炎疫苗的研究进展。  相似文献   

6.
乙型脑炎疫苗研究进展   总被引:3,自引:0,他引:3  
乙型脑炎(乙脑)的流行是世界性的公共卫生问题之一,其流行范围正逐步扩大。目前对于乙脑尚无特效治疗手段,因此预防乙脑的发生尤为重要。随着对日本脑炎病毒研究的深入,利用基因工程技术研制新型候选疫苗已成为预防乙脑新的发展方向。我们简要综述了乙型脑炎病毒的基因组结构及其蛋白功能,以及国内外乙型脑炎疫苗研发的新进展。  相似文献   

7.
Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.  相似文献   

8.
Wang  Ran  Xie  Lyu  Gao  Na  Fan  Dongying  Chen  Hui  Wang  Peigang  Zhou  Hongning  An  Jing 《中国病毒学》2019,34(3):243-252
The incidence of Japanese encephalitis(JE) has significantly decreased in China due to JE vaccines. In this study, we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV) to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children. A total of 300 serum samples were collected from vaccinated children aged 3–10 years in Zhaotong, Yunnan,China. The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test. The highest seropositivity of 82% was observed in vaccinated children during the first0.5–1.5 years after booster vaccination. Then, the seropositivity began to decline and remained lower than the original level observed in the 0.5–1.5-year group. An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination. Similarly, the neutralizing antibody(nAb) titres gradually decreased over time, and the levels showed a positive correlation with the protective efficacy in mice. This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine. Our results provide data that clarify the persistence and waning of antibodies to JEV, which may help elucidate the pathogenesis of JE.  相似文献   

9.
Xie  Shengda  Liang  Zhenjie  Yang  Xingmiao  Pan  Junhui  Yu  Du  Li  Tongtong  Cao  Ruibing 《中国病毒学》2021,36(6):1503-1519
Virologica Sinica - Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a...  相似文献   

10.
中国流行性乙型脑炎病毒分子生物学特性研究   总被引:39,自引:0,他引:39  
为了从分子水平了解中国流行性乙型脑炎(乙脑)病毒与国外分离株的差异,对1949年以来在中国乙脑主要流行地区分离的19株乙脑病毒的PrM-C区及E蛋白基因区的核苷酸序列进行了对比研究,以期了解不同时间不同地区在中国流行的乙脑病毒的分子差异.结果显示:病毒生物学初步鉴定显示,病毒感染BHK细胞后56h所有细胞均发生病变,约50%以上细胞脱落(CPE( ));3日龄乳鼠接种病毒72h之内死亡;所有病毒均与乙脑病毒(A2株)抗血清发生阳性反应,但反应强度不同,提示所有病毒均为乙脑病毒.病毒PrM-C(456~695)区核苷酸序列与各个国家及地区、各个基因型以及各个年代的73株乙脑病毒相应序列,用Woan-Ru Chen建立的方法进行基因分型分析,结果显示,所有19株中国分离的病毒均属于基因型Ⅲ型,与国外相关文献所报道的乙型脑炎病毒中国分离株的基因型相符.以乙型脑炎病毒疫苗株P3株为标准,对各病毒E蛋白500个氨基酸序列进行分析.各毒株核苷酸差异在0和4.2%之间,氨基酸差异在0和4.0%之间.所有核苷酸差异均为碱基的替换,无论核苷酸或氨基酸均无插入或丢失.大多数核苷酸变异在氨基酸编码的第三位,属于沉默突变,不引起氨基酸变化.18株病毒E蛋白活性结构域与疫苗株P3株相比共有247个氨基酸的差异,平均每株病毒有12.35个氨基酸的差异,有11株病毒(SA14、TLA、CZX、CBH、G35、GSS、LYZ、SH-3、YLG、YN、ZMT)在该区的差异数低于这个平均值.而在超过这一平均数的7个病毒株(A2、HA-3、47、CH-13、CTS、LFM、ZSZ)中,LFM、ZSZ、47与疫苗株相比较分别有16~20个氨基酸的差异,相对其它17株病毒而言,差异较为明显.以上线索提示,现有的乙型脑炎灭活疫苗在理论上可以覆盖包括现存病毒在内的毒株.  相似文献   

11.
黄莺  刘珊  杨鹏  杜韫  孙志伟  俞炜源 《生物工程学报》2009,25(10):1532-1537
为了表达日本脑炎病毒囊膜蛋白(E蛋白)结构域DⅢ区,了解其作为亚单位疫苗的可能性,本研究根据SA14-14-2病毒株序列(GenBank Accession No.D90195)设计两条引物,以全长JEV感染性克隆pBR-JTF为模板,通过PCR扩增出JEVE蛋白DⅢ的cDNA片段,构建了原核表达载体pET-JEDⅢ,转化大肠杆菌Rosetta(DE3)进行融合表达。融合蛋白为可溶性表达,表达量约占菌体蛋白的75%。用纯化后蛋白免疫新西兰兔和BALB/C鼠,通过ELISA,Western blotting,噬斑减少实验,及乳鼠攻毒实验验证JEDⅢ的抗原性和免疫原性。Western blotting及ELISA结果表明纯化后的表达产物具有良好的抗原性,纯化的JEDⅢ蛋白免疫新西兰兔,可以获得高达1:7×105滴度的抗JEV特异性抗体;JEDⅢ蛋白免疫BALB/C鼠,可以获得1:8.2×104滴度的抗JEV特异性抗体。并且获得1:256滴度的中和抗体,乳鼠攻毒实验能达到75%的保护效果。以上结果说明本研究表达、纯化的重组JEDⅢ蛋白,免疫小鼠以及兔后,能产生抗JEV的特异性抗体,中和性抗体,能够保护部分乳鼠接受毒...  相似文献   

12.
Skin-resident dendritic cells (DCs) likely encounter incoming viruses in the first place, and their migration to lymph nodes following virus capture may promote viral replication. However, the molecular mechanisms underlying these processes remain unclear. In the present study, we found that compared to cell-free viruses, DC-bound viruses showed enhanced capture of JEV by T cells. Additionally, JEV infection was increased by co-culturing DCs and T cells. Blocking the C-type lectin receptor DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) with neutralizing antibodies or antagonists blocked JEV transmission to T cells. Live-cell imaging revealed that DCs captured and transferred JEV viral particles to T cells via virological synapses formed at DC-T cell junctions. These findings indicate that DC-SIGN plays an important role in JEV transmission from DCs to T cells and provide insight into how JEV exploits the migratory and antigen-presenting capabilities of DCs to gain access to lymph nodes for dissemination and persistence in the host.
  相似文献   

13.
Japanese encephalitis is a mosquito borne disease and is the leading cause of viral encephalitis in the Asia-Pacific area. The causative agent, Japanese encephalitis virus(JEV) can be phylogenetically classified into five genotypes based on nucleotide sequence. In recent years, genotype I(GI) has displaced genotype III(GIII) as the dominant lineage, but the mechanisms behind this displacement event requires elucidation. In an earlier study, we compared host variation over time between the two genotypes and observed that GI appears to have evolved to achieve more efficient infection in hosts in the replication cycle, with the tradeoff of reduced infectivity in secondary hosts such as humans. To further investigate this phenomenon, we collected JEV surveillance data on human cases and, together with sequence data, and generated genotype/case profiles from seven Asia-Pacific countries and regions to characterize the GI/GIII displacement event. We found that, when comprehensive and consistent vaccination and surveillance data was available, and the GIII to GI shift occurred within a well-defined time period, there was a statistically significant drop in JEV human cases. Our findings provide further support for the argument that GI is less effective in infecting humans, who represent a dead end host. However, experimental investigation is necessary to confirm this hypothesis. The study highlights the value of alternative approaches to investigation of epidemics, as well as the importance of effective data collection for disease surveillance and control.  相似文献   

14.
使用2×107PFU乙脑病毒野毒株GSS抗原preM-E-NS1-NS2a的非复制型重组痘苗病毒NTV-JEV免疫3周龄BALB/c小鼠,小鼠体内乙脑病毒特异性抗体滴度1∶71,IgG2a/IgG1为1.5,中和抗体滴度为1∶8,免疫组小鼠产生特异性补体介导的细胞杀伤作用。以10 LD50乙脑P3株病毒对小鼠进行了脑内攻击,NTV-JEV免疫组小鼠存活率为100%,与乙脑减毒活疫苗SA14-14-2免疫组存活率相同。100 LD50P3株病毒攻击后NTV-JEV免疫组存活率28.6%,与SA14-14-2组免疫效果无明显差别。存活小鼠体内乙脑病毒特异性抗体升高为1∶179,IgG2a/IgG1比值为1.9,中和抗体大于1∶16。以30 LD50GSS株病毒进行攻击后,NTV-JEV免疫组和SA14-14-2组保护率均为8.3%。上述结果表明,NTV-JEV与乙脑减毒活疫苗SA14-14-2株具有相近的保护力,NTV-JEV不仅可以保护小鼠抵抗同株病毒GSS的攻击,而且可以抵抗异株病毒P3的攻击。  相似文献   

15.
Japanese encephalitis(JE) is a viral encephalitis disease caused by Japanese encephalitis virus(JEV) infection. Uncontrolled inflammatory responses in the central nervous system(CNS) are a hallmark of severe JE. Although the CCR2–CCL2 axis is important for monocytes trafficking during JEV infection, little is known about its role in CNS trafficking of CD8~+T cells. Here, we characterized a mouse model of JEV infection, induced via intravenous injection(i.v.) and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice. The CNS expression of chemokines, Ccl2, Ccl3, and Ccl5, and their receptors, Ccr2 or Ccr5, was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis. Moreover, JEV infection resulted in the migration of a large number of CD8~+T cells into the CNS. In the brains of JEV-infected mice, infiltrating CD8~+T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells(CD44~+CD62 L~-). JEV infection dramatically enhanced the expression of programmed death 1(PD-1) on infiltrating CD8~+T cells in the brain, as compared to that on peripheral CD8~+T cells in the spleen. This effect was more pronounced on infiltrating CCR2~+CD8~+T cells than on CCR2-CD8~+T cells. In conclusion,we identified a new subset of CD8~+T cells(PD1~+CCR2~+CD8~+T cells) present in the CNS of mice during acute JEV infection. These CD8~+T cells might play a role in JE pathogenesis.  相似文献   

16.
Yu  Du  Zhao  Yundi  Pan  Junhui  Yang  Xingmiao  Liang  Zhenjie  Xie  Shengda  Cao  Ruibing 《中国病毒学》2021,36(6):1443-1455
Virologica Sinica - The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1′ produced through a programmed -1...  相似文献   

17.
Li  Mengyuan  Yang  Jiali  Ye  Chuantao  Bian  Peiyu  Yang  Xiaofei  Zhang  Haijun  Luo  Chuanyu  Xue  Zhifeng  Lei  Yingfeng  Lian  Jianqi 《中国病毒学》2021,36(6):1554-1565
Virologica Sinica - Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis in endemic regions of Asia. The neurotropism of JEV and its high-efficiency replication in neurons are...  相似文献   

18.
为探索包膜蛋白T120A突变对乙脑/寨卡嵌合病毒JEV/ZIKV小鼠脑内神经毒力的影响,应用重叠延伸PCR和分子克隆技术构建含T120A突变的乙脑/寨卡嵌合病毒全长cDNA质粒,经酶切、测序鉴定后,以其为模板体外转录制备RNA,电转染导入BHK21细胞,收获培养液上清获得突变病毒株JEV/ZIKV (T120A).分别...  相似文献   

19.
在以往构建的JEV疫苗株SA14-14-2感染性克隆pBRkpn-JTF的基础上,构建了去除JEV结构区域基因(prM基因和E基因),保留非结构区域基因的复制子表达载体pCTCJEV、pCTMJEV,转染BHK-21细胞后,通过反转录PCR(RT-PCR)和间接免疫荧光实验(IFA)检测该载体的自主复制能力和蛋白表达情况,结果证实该载体在BHK-21细胞中能够自主复制并且表达非结构蛋白。进一步在该载体中插入报告基因EGFP,检测外源蛋白的表达情况,结果显示在转染复制子载体的24h后,荧光显微镜下能够看见绿色荧光蛋白的表达,阳性信号持续增强,能够维持10d左右,证实了构建的复制子载体pCTCJEV能够有效地表达外源蛋白,这为进一步建立以乙脑病毒复制子为基础的疫苗载体系统打下基础。  相似文献   

20.
目的:制备乙型脑炎病毒(JEV)可视化分型基因芯片。方法:根据JEV的基因组序列,应用生物学软件设计JEV分型引物及探针,制备其可视化分型基因芯片;用生物素标记的引物PCR扩增目的片段,并与固定于玻片上的探针杂交,加入链霉亲和素标记的纳米金,银增强实现可视化;进行特异性、灵敏性及重复性试验。结果:探针特异地与相应的标记目的基因片段杂交,并在芯片上呈现较强的阳性杂交信号;2号探针能特异性检出JEV,3、4号探针可分别对Ⅰ型和Ⅲ型JEV进行分型;芯片对JEV质粒检测的灵敏度达105拷贝/mL;以蓝耳病病毒等5种病毒为对照,芯片只对JEV响应,具有特异性;制备的基因芯片具有批间、批内重复性。结论:制备的基因芯片具有高特异性、灵敏性及重复性,可以快速、准确、高通量地对JEV进行可视化分型检测。  相似文献   

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