人偏肺病毒黏附蛋白的二级结构及B细胞表位初步预测

目的预测人偏肺病毒G蛋白的二级结构及B细胞表位。方法分别采用SOPMA方法及HMMTOP程序预测G蛋白的二级结构和跨膜区域；综合分析蛋白的柔性结构、亲水性、表面可及性与抗原性指数，预测G蛋白的抗原表位。结果G蛋白的二级结构主要为柔性区域，占62．1％；廿螺旋占22．37％；β-折叠占15．53％；N端第32～51位氨基酸残基为跨膜区。B细胞表位位于G蛋白N端55—77、80-104、111～126、130～167、178—210区段。结论应用多参数预测G蛋白的二级结构与B细胞表位，为进一步研究蛋白特征、单克隆抗体制备及表位疫苗研制奠定了基础。     

人类偏肺病毒F蛋白的B细胞表位预测

目的 预测人类偏肺病毒F蛋白的B细胞表位。方法综合分析二级结构、亲水性、表面可及性与抗原性指数，预测F蛋白的抗原表位。结果B细胞表位位于F蛋白N段15-28、88-102、161-189、195-202、206-213、245-257、290-299、302-309、318-334、410-419、515-526区段。结论应用多参数预测F蛋白的B细胞表位，为进一步研究蛋白特征、单克隆抗体制备及表位疫苗研制奠定了基础。     

西尼罗病毒多表位基因的融合表达及其免疫保护作用研究

目的研究西尼罗病毒（WNV）多表位基因的融合表达及其免疫保护作用。方法利用生物信息软件Biosun分析西尼罗全基因序列，确定表位基因片段并选择合适片段连接构成多表位基因。采用重叠PCR方法扩增该基因，构建多表位基因的原核重组表达载体pET-43a-M，进行融合表达和纯化。将表达蛋白加弗氏佐剂免疫小鼠并进行攻毒试验，观察其保护作用。结果分子克隆和构建了原核重组表达质粒pET-43a-M，表达和纯化了融合蛋白Nus-M，该蛋白免疫后的小鼠能够部分抵御西尼罗病毒的攻击。结论构建的西尼罗病毒多表位基因能够在原核细胞内表达，表达产物有较弱的免疫保护反应，为该病毒多表位抗原的串联及多表位疫苗研究提供了试验资料，但需要进一步改进。     

北京地区人偏肺病毒核蛋白基因的原核表达及抗原活性分析

目的用大肠杆菌表达获得人偏肺病毒主要结构蛋白N蛋白，为下一步的深入研究奠定基础。方法从重组质粒pUCm-N1816中PCR扩增获得N基因，用BamHⅠ和EcoRⅠ双酶切后插入到原核表达载体pET30a（＋）中，得到重组表达质粒pE130a-N1816，转化大肠杆菌BL21（DE3），IPIG诱导培养。SDS-PAGE和Western blot检测目的蛋白的表达和抗原性。结果经双酶切和测序证明1．2kb N1816。基因正确插入pET30a中，并具有正确的读码框架，得到预期的pET30a-N1816重组原核表达质粒。37℃，1mmol／L IFIG诱导培养6h后产生大量带6个组氨酸标记的重组N蛋白，主要以包涵体形式存在，占细胞总蛋白的20％左右。N蛋白粗提物经Co^2＋亲和层析获得较理想纯化。Western blot结果显示，体外所表达的蛋白能和特异性抗血清及人血清反应。结论本研究成功构建了重组pET30a-N1816原核表达质粒，N蛋白获得了高效表达，并且具有特异抗原活性，可用于人偏肺病毒的深入研究。     

绿色荧光蛋白标记重组人偏肺病毒的制备

目的 人偏肺病毒(human metapneumovirus,hMPV)可致人上、下呼吸道感染.研究利用反向遗传学技术,以带绿色荧光蛋白(GFP)标记的hMPV NL/1/00全长cDNA质粒及4种辅助质粒pCITE-N、pCITE-P、pCITE-M2.1和pClTE-L为基础,在体外制备GFP标记的重组hMPV病毒(命名为rhMPV NL/1/00 GFP).方法 采用LipofectAMINE 2000将带GFP标记的hMPV NL/1/00全长cDNA质粒以及蛋白表达质粒pcITE-N、pCITE-P、pCITE-M2.1和pcITE-L共转染BSR-T7细胞,3 d后取BSR-T7细胞上清液感染Vero-E6细胞,1-4 d后观察Veto-E6细胞出现明显细胞病变(CPE)和绿色荧光,持续观察至第10天.收集病毒上清用于病毒滴度检测.提取培养上清的病毒RNA并通过RT-PCR方法扩增病毒N基因、F基因和G基因验证所获重组病毒.结果 Vero-E6细胞接种1～4 d后观察到明显CPE和绿色荧光,随后CPE加重,荧光信号加强,持续至感染后10 d;第1、5、10、15和20代病毒的滴度波动于105.0～106.5TCID50/ml;RT-PCR检测出符合预期大小的910 bp(N)、450 bp(F)和980 bp(G)条带,经卜述片段cDNA序列测定和比对表明获得的重组病毒与hMPV NL/1/00原型病毒序列一致.rhMPV NL/1/00 GFP重组病毒在体外传代20代后,遗传信号和荧光信号均稳定.结论 采用反向遗传学技术成功拯救了具有感染性的重组hMPV病毒,为hMPV感染发病机制及疫苗研究奠定了基础.     

人偏肺病毒研究进展

<正>人偏肺病毒(human metapneumovirus,hMPV)是新近发现的一种呼吸道致病病毒,2001年首次在荷兰一婴儿的鼻咽部抽吸物中被分离得到,根据它的形态学、生物化学以及基因学特点,hMPV一开始被分类为禽偏肺病毒,禽偏     

绿色荧光标记重组人偏肺病毒空斑形成滴度测定方法的建立

目的 建立用于绿色荧光标记的重组人偏肺病毒(GFP-rhMPV)空斑形成滴度测定方法.方法 基因组中插入绿色荧光蛋白基因的hMPV全序列cDNA质粒和主要蛋白质表达质粒转入细胞293T后获得感染性重组hMPV.GFP-rhMPV在Vero-E6细胞中连续传代提升病毒滴度并保存.将等倍稀释的重组病毒液接种常规制备的Vero-E6细胞单层,用含或不含胰酶的低熔点琼脂精凝胶覆盖细胞,孵育一定时间后,采用荧光显微镜下计数荧光空斑数和抗原抗体蓝斑形成法计算病毒滴度.结果 感染后3 d,荧光显微镜下GFP-rhMPV可在低熔点琼脂糖凝胶覆盖层下形成分界较为清晰的绿色荧光集落,接种后3 d荧光空斑相对独立,便于计数.蓝斑形成法在感染后第5天蓝斑较大,易观察.此前拯救获得的GFP-rhMPV在宿主细胞Vero-E6中的复制滴度可达1×106 PFU以上.结论 成功建立了GFP-rhMPV的空斑形成实验滴度定量检测方法,为hMPV的致病机制、防治手段研究奠定了基础.     

汉滩病毒部分核蛋白基因原核表达产物 的免疫及其保护作用

目的 探讨汉滩病毒（HTNV）部分核蛋白（NP）基因（37－294bp)原核表达产物NP AA1－86多肽的免疫原性及其免疫保护作用。方法 大肠杆菌表达的HTNV部分核蛋白多肽NP AA1－86混合福氏佐剂,腹腔注射免疫BALB／c小鼠,IFA检测抗体应答;^3H-TdR掺入及4h ^51Cr释放试验检测免疫小鼠脾细胞对HTNV的特异淋巴细胞增殖转化指数及细胞毒T淋巴细胞（CTL）杀伤功能;免疫小鼠脾细胞转输感染HTNV A9株的乳鼠,观察对乳鼠致死性的免疫保护作用。结果 免疫后1周小鼠即出现抗体应答反应,抗体滴度最高达1：12800,与重组完整NP免疫组差异无显著性（P＞0．05）;免疫小鼠的淋巴细胞增殖转化指数、CTL杀伤率较对照组明显增高（P＜0．01）,与完整NP免疫组差异无显著性（P＞0．05）,CTL杀伤率随效：靶比例的增高呈逐渐上升趋势;免疫小鼠脾细胞转输可部分保护病毒致死性感染的乳鼠,保护率约25％。结论 大肠杆菌表达的汉滩病毒部分核蛋白多肽NP AA1－86不仅包含NP几乎所有的体液免疫表位,同时含有部分细胞免疫表位,对汉滩病毒感染可产生部分免疫保护作用。     

人偏肺病毒A与B基因型对小鼠致病性的观察

目的 观察人偏肺病毒( human metapneumovirus,hMPV)A和B亚型感染BALB/c小鼠后,小鼠在体重、病毒载量、病理及气道反应性等方面的改变,探讨两亚型病毒致病性上的差异,为hMPV的进一步研究提供基础资料.方法 hMPV感染BALB/c动物模型后,通过使用real-time RT-PCR方法检测鼠肺内病毒载量的变化,肺功能检测仪监测小鼠气道反应性的变化及组织系统评分判定病理改变情况,来观测两亚型致病性的差异.结果 hMPV两亚型感染组,在体重的动态监测、病毒载量、肺组织病理改变及气道反应性上差异均无统计学意义.结论 hMPV两基因型感染BALB/c小鼠的致病性无差异.     

人偏肺病毒病原学与防治研究进展

人偏肺病毒（human metapneumovirus,HMPV）是2001年由荷兰学者vanden Hoogen等[1]首次从儿童呼吸道感染标本中分离发现的.研究发现其在世界范围内均有流行,可引起呼吸道感染,尤其在儿童、患有基础疾病的成人、老年人、免疫功能不全或器官移植患者可引起重症呼吸道感染.目前针对HMPV尚没有特异的抗病毒药物及安全有效的疫苗.本文就目前该病毒的病毒学特征、实验室诊断、治疗及疫苗研制的研究现状进行综述.     

乙型肝炎病毒多表位抗原DNA疫苗的免疫原性

目的 :探讨HBV复合多表位DNA疫苗诱导体液及细胞免疫的可行性。方法 :将HBV多表位抗原基因BPT克隆到真核表达载体pcDNA3.1中 ,构建重组真核表达载体pcDNA3.1/BPT。将其通过肌肉注射免疫BALB/c小鼠 ,用间接免疫ELISA法、CTL杀伤功能检测和淋巴细胞增殖试验 ,检测特异性体液免疫和细胞免疫的水平 ,并观察其对免疫小鼠的毒副作用。结果 :用重组质粒pcDNA3.1/BPT免疫BALB/c小鼠后 ,在效靶比为 10 0∶1时 ,可诱导显著地特异性CTL应答 (P <0 .0 5 )。ELISA检测免疫小鼠血清特异性IgG的水平明显升高 (P <0 .0 5 )。在BPT基因原核表达蛋白的刺激下 ,免疫小鼠脾淋巴细胞增殖显著 (P <0 .0 5 )。RT PCR分析表明 ,IL 12mRNA的水平亦明显升高。结论 :HBV多表位基因疫苗可诱发特异性免疫应答 ,为预防、治疗性HBV疫苗的研制提供了一定的实验依据     

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.     

Seroprevalence of human metapneumovirus (hMPV) in the Canadian province of Saskatchewan analyzed by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay

Human metapneumovirus (hMPV) is a newly identified respiratory virus associated with respiratory tract infection in both adults and children. Previous reports showed that infection of hMPV appeared to be ubiquitous. To determine the seroprevalence of hMPV, a total of 576 human sera from patients in Saskatchewan, Canada, were screened by enzyme-linked immunosorbent assay (ELISA) based on expression of the nucleocapsid (N) protein of hMPV in recombinant baculovirus. The recombinant N protein with a molecular mass of 43.5 kDa was abundantly produced in insect cells. Moreover, the recombinant N proteins of the prototype viruses for the two major groups of hMPV have cross-antigenicity. The seropositive rate for each age group was 13.5% (13/96) (0-5 years), 26.1% (25/96) (6-10 years), 32.3% (31/96) (11-15 years), 99.0% (95/96) (16-30 years), 91.7% (88/96) (31-60 years), and 93.8% (90/96) (61+ years), respectively. The data indicated that exposure to hMPV is a common phenomenon. The ELISA based on recombinant baculovirus produced N protein of hMPV provides a useful tool for seroepidemiological study of this virus.     

带有HBV基因的腺相关病毒为载体的重组病毒感染树突状细胞的研究

目的探讨腺相关病毒（Adeno-associated virus，AAV）为载体的含有HBV—S、C或X基因的重组病毒（rAAV—HBV—S，C，X）感染正常人树突状细胞的效率。方法分别构建含有HBV-S、C和x基因的从v质粒，在293细胞中包装成重组病毒，测定病毒滴度。从正常人外周血中分离出单核细胞进行体外培养，在培养的第1天分别用rAAV—HBV—S、C、X和作为对照的293细胞裂解液感染刺激单核细胞，感染后的单核细胞在GM-CSF、IL-4和TNF-α作用下继续培养7d获得成熟的树突状细胞。用RT-PCR及流式细胞仪细胞内染色检测HBV基因的RNA和蛋白的表达。结果重组rAAV—HBV—S、C、X的病毒滴度可以达到10^7拷贝／ml，用重组病毒分别感染树突状细胞。与对照组相比，感染的树突状细胞可表达相应的HBV基因RNA，流式细胞仪检测显示约90％的树突状细胞有相应HBV蛋白的表达。结论rAAV-HBV可有效感染树突状细胞，提示可以通过该途径刺激树突状细胞提呈抗原。     

Induction of T cell response by bluetongue virus core-like particles expressing a T cell epitope of the M1 protein of influenza A virus

A CD4⁺ T cell epitope of the influenza virus matrix protein corresponding to the C terminus (QAYQKRMGVQMQRFK) was inserted into the VP7 gene of bluetongue virus (BTV). The chimeric protein was expressed by a dual recombinant Autographa californica polyhedrosis virus (AcNPV), which encodes the two inner capsid proteins VP3 and VP7 of BTV. When Spodoptera frugiperda cells (Sf9 cells) were infected with this recombinant BTV, core-like particles (CLPs) were formed as demonstrated by electron microscopy. To study the immunogenicity of a foreign epitope deprived of its natural flanking sequences in vitro, purified CLPs expressing the T cell epitope were used to stimulate two different MHC class II-restricted CD4⁺ human T cell clones. One of these T cell clones, ALF 3.7 was specific for the inserted epitope, whereas the other T cell clone ALF 4.4 recognized shorter derivates of the given epitope. CLPs with the inserted epitope were presented as efficiently as purified influenza virus matrix protein to the clone ALF 3.7, whereas clone ALF 4.4 showed no proliferative response. Received: 17 February 1998     

人源性抗HFRS病毒抗体基因真核表达载体的构建及鉴定

构建人源性抗ＨＦＲＳ病毒基因工程抗体基因的表达载体,为其进一步在真核细胞中表达奠定基础。方法：经多次克隆将人源性抗ＨＦＲＳ病毒抗体可变区基因与启动子,增强子、前导肽序列和剪接供体信号等真有达元件及人抗体恒定区基因和抗生素选标记基因相连接。     

Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are important respiratory pathogens of small children and adults. The present study aimed to design a sensitive real-time RT-PCR assay for the detection of hRSV and hMPV in comparison with direct fluorescent assay (DFA) and to determine the incidence of hMPV and hRSV as causative agents of respiratory infections in a Finnish population. For DFA detection of hMPV antigen, four commercial antibodies were evaluated. The duplex real-time RT-PCR assay achieved a sensitivity of 10³ copies/mL of specimen for hRSV and hMPV type A viruses and 10⁴ copies/mL for type B hMPV. The detection rate of the RT-PCR assay was compared with those for DFA detection of hMPV and hRSV in analyses of 350 nasopharyngeal aspirates sent to HUSLAB, Helsinki University Hospital, for routine virus diagnostics during November 2007 to June 2008. Of the samples analyzed, 43 (12.3%) were positive for hRSV by DFA and an additional 13 specimens (3.7%) were positive for hRSV by RT-PCR. Only four samples (1.1 %) were found to be positive for hMPV RNA by RT-PCR, with two of them also positive by DFA. The duplex real-time RT-PCR assay described in the present study can therefore be applied for efficient identification of hMPV and hRSV in clinical specimens and collection of information on the epidemiology and clinical outcome of these viruses.     

土拨鼠肝炎病毒核心蛋白的高效表达和B细胞表位鉴定

目的 建立高效表达土拨鼠肝炎病毒核心蛋白（WHcAg）的方法并对其B细胞表位进行鉴定。方法分别构建不同截短型WHcAg的质粒以了解能大量表达并形成WHV核心颗粒的区段，利用变性WHcAg制备单克隆抗体以寻找能与WHcAg线性B细胞表位结合的单克隆抗体。结果 WHcAg前144或149氨基酸残基能够大量表达并能形成颗粒样结构。这2种截短型WHcAg能够在大肠杆菌中表达并组装成直径为34肿的核心颗粒，最终纯化获得毫克级的核心蛋白。截短型WHcAg保留了全长WHcAg的抗原性，而且变性WHcAg存在另外的B细胞线性表位，利用变性WHcAg进行免疫制备单克隆抗体获得的5株抗体均针对WHcAg的N末端表位，该表位在WHcAg和HBcAg高度保守。结论 建立了高效表达WHcAg的方法，制备了针对WncAg单克隆抗体。并对WHcAg的线性B细胞表位进行了鉴定，发现WHcAg和HBcAg的N末端存在共同的线性B细胞表位。     

Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus

We report on the induction of primary and long-term memory cytotoxic T lymphocyte (CTL) responses against the nucleoprotein of the influenza virus A/PR8/34 in mice immunized with plasmid DNA targeted to B lymphocytes in the spleen. We found that the magnitude of the CTL response and the size of the pool of memory CTL was greater when the CTL response was induced in presence of T cell help. Interestingly, immunization with a signal sequence-competent transgene was markedly superior to immunization with a transgene lacking the endoplasmic reticulum (ER) targeting sequence, in inducing CTL. We also found a correlation between in vivo protection from lethal virus challenge and (1) the availability of T cell help and (2) ER targeting. Immunization of dendritic cell-deficient mice suggests that B lymphocytes function as antigen-presenting cells in this model of immunization. Collectively, the results suggest that somatic transgene immunization is a conceptually new approach to induce effective anti-viral CTL responses and to assess the parameters critical for long-lasting and protective CTL responses in vivo.