我国常见钩端螺旋体蛋白质免疫化学及免疫生物学特性…

分别用我国常见的１５群１５型钩端螺旋体多克隆抗体对我国常见的１５群１５型钩端螺旋体全菌蛋白进行了Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ分析。结果显示：用不同群的多克隆抗体与１５群１５型钩端螺旋体参考株的全菌蛋白反应，其图谱并不相同，表现在阳性反应带数量的多少和分子量的变化；而用同一群多克隆抗体与１５群１５型钩端螺旋体参考株的全菌蛋白反应，其Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ图谱非常相似，都有３条分子量分别     

我国常见钩端螺旋体蛋白质特性的研究

应用SDS-PAGE首次对我国常见的15群15型钩端螺旋体参考株的全菌蛋白进行了分析,发现15群15型参考株的全菌蛋白基本由33条谱带组成,其中主要蛋白谱带16条,它们的分子量分别为15500,16800,18000,23000,25000,29500,32000,39000,45000,70000,80000,84000,100000,110000,114000和128000;次要蛋白谱带17条,它们的分子量分别为     

乳糖发酵奈氏菌和脑膜炎奈氏菌诱生的流脑杀菌抗体…

利用杀菌力试验测定不同来源血清对脑膜炎奈氏菌（Ｎｍ）的杀菌抗体水平。结果表明Ａ群流脑病人恢复期血清、带菌者及乳糖发酵奈氏菌（Ｎ１）带菌者血清／兔抗Ｎｍ及Ｎ１血清中均具有对Ｎｍ的杀菌抗体。通过Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ分析发现上述人血清ＩｇＧ、ＩｇＭ和ＩｇＡ均与这两种奈氏菌的５２ｋＤ、１、２或３类外膜蛋白发生特异反应。用纯化的１和３类外膜蛋白及脂寡糖免疫小鼠制备免疫血清，测定抗体水平表明，Ｎ１     

问号钩端螺旋体铁离子调节蛋白A的免疫原性及保守性研究

目的克隆表达和鉴定问号钩端螺旋体（L．interrogans，简称钩体）黄疸出血群赖型赖株中铁离子调节蛋白A（IRPA），研究IRPA的免疫原性和在不同钩体菌种中的保守性，探讨其在致病和疫苗研究中的意义。方法生物信息学软件分析预测IRPA的特征。构建原核表达质粒pQE31-IRPA，经IPTC诱导后用SDS-PAGE及Western blot鉴定表达情况。用表达的重组蛋白免疫BALB／c小鼠，Western blot检测其免疫原性和在不同血清型钩体中的保守性。ELISA和Western blot检测钩体全菌兔抗血清中的IRPA抗体。结果生物信息学预测结果显示，IRPA是可能位于外膜的脂蛋白，在钩体内有多个蛋白可能与之相互作用。成功克隆表达了重组质粒pQE31-IRPA，重组蛋白能刺激BALB／c小鼠产生抗体（效价为1：32000），并能与相应抗体反应，具有良好的免疫原性。在15株不同血清型的问号钩体及1株双曲钩体（L．biflex）中均可检测到重组蛋白的表达，并在钩体全菌兔抗血清中检测到其抗体。结论IRPA具有良好的免疫原性和保守性，为进一步研究其在钩体病中的作用机制及其作为候选基因疫苗的研究奠定了基础。     

B群脑膜炎奈瑟氏菌外膜蛋白抗原性分析

挑选了１０株具有代表性的菌株，提取了它们的外膜蛋白（ＯＭＰｓ），ＳＤＳ－ＰＡＧＥ分析发现：１０株菌的ＯＭＰｓ带谱不完全相同，但都含有１，２／３，４和５类外膜蛋白，而且纯化的１类外膜蛋白（ＯＭＰ１）显示一条分子量为（４１或４３）×１０＾３的蛋白带。从中选两株菌５４２８５２（Ｂ：ＮＴ：Ｐ１．２：Ｌ３，７，９：克隆Ｉ：ＲＦＬＰ－ｂ２０）和３４０７（Ｂ：１５：Ｐ１．２：Ｌ３，７，９，克隆群Ｉ：ＲＦＬＰ－ｂ     

空肠弯曲菌HSP60家庭成员的鉴定

用针对ＨＳＰ６０保守区的二株单抗ＩＬＨ９和ＭＬ－３０进行了Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ和ＥＬＩＳＡ试验，发现空肠弯曲菌ＣＪ－Ｓ１３１株４３ｋＤ通道蛋白属ＨＳＰ６０家庭成员，命名其为空弯菌ＨＳＰ４３。ＳＤＳ－ＰＡＧＥ和薄层扫描显示ＨＳＰ４３是主要菌体蛋白。虽然ＨＳＰ极端保守，但用ＣＪ－Ｓ１３１ｌｙｓａｔｅ免疫小鼠事，发现最早诱导的抗体是针对ＨＳＰ４３的，并且这种抗体活性在随后８０ｄ的观察期间一直     

钩端螺旋体内毒素的实验研究 Ⅴ 钩端螺旋体脂多糖增强机体特异性抗感染能力的作用

波摩那群波摩那型56608株钩端螺旋体脂多糖(L-LPS)免疫的豚鼠能抵抗同株钩端螺旋体的攻击而免于发病和死亡,旦其心血和肾脏培养阳性率也明显下降。L-LPS免疫动物后可产生以IgM为主的群特异性抗体。该抗体当有补体存在时,能在体外裂解钩端旋螺体。此外,L-LPS尚能特异性地抑制豚鼠腹腔巨噬细胞的粘附作用。实验结果提示L-LPS为一种保护性抗原,可特异性地增强机体抗感染的能力。     

钩端螺旋体DNA限制性内切酶图谱鉴定分类的初步研究

用ＥｃｏＲⅠ等六种限制性内切酶消化分析了七日热群和波摩那群各型标准株以及一些野生株钩端螺旋体ＤＮＡ。结果表明，不同血清型菌株具有完全不同的限制性内切酶图谱（ＲＥＰ）；来自不同地域、不同时期、不同宿主的同型野生株ＲＥＰ一致，与它们相应的标准株ＲＥＰ没有差别或仅有微小差别。说明钩端螺旋体ＤＮＡ内切酶图谱具有血清型特征性ＲＥＰ，而且相当稳定。本研究测算出七日热和波摩那各型标准株的ＥｃｏＲⅠＲＥＰ五条高分子带的分子量，并以此作为标准鉴定了部分野生苗株。结果显示，仅凭ＥｃｏＲⅠＲＥＰ两条最大的带就可鉴别两群各型菌株。我们认为ＲＥＰ是一种比较灵敏、快速、简便、准确的钩端螺旋体分类方法，所分型别与传统的血清学型别基本一致。     

抗恶性疟HRP—Ⅱ单链抗体的筛选和鉴定

目的 研究开发简便、快速和有铲的疾诊断技术。方法 利用菌体抗体库技术，在构建抗恶性疟原虫红内期噬菌体抗加的基础上，经３轮“吸附－洗脱－扩增”的富集反应后，筛选抗ＨＲＰⅡ阳性克隆株并进行可溶性诱导表达，最后用ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ等进行鉴定。结果 筛选出６株抗ＨＲＰⅡ阳性克隆株表达的单链抗体Ｍｆ为３１０００左右，能与ＨＲＰⅡ抗原起特异性结合反应。结论 本研究为研制疟疾快速诊断试剂盒奠定     

问号钩端螺旋体血清群LipL32基因型分析及其重组蛋白的免疫学鉴定

目的　探讨 15群 15型问号钩端螺旋体 (简称钩体 )中国参考标准株及 2群 2型双曲钩体国际参考标准株是否均存在主要外膜蛋白 (MOMP)基因 (LipL32 ) ,克隆并构建该基因的原核表达系统 ,鉴定表达产物的免疫性。方法　常规酚 氯仿法提取上述钩体株基因组DNA ,高保真PCR扩增全长LipL32基因片段 ,T A克隆后测定核苷酸序列并构建表达系统 ,不同浓度IPTG诱导后用SDS PAGE检测rMOMP表达情况。分别用兔抗TR patocⅠ钩体全菌抗血清、rMOMPs免疫兔血清的Westernblot鉴定其免疫反应性和免疫原性 ,显微镜凝集试验 (MAT)检测rMOMPs免疫兔血清的交叉凝集效价 ,钩体细胞黏附模型检测抗体阻断效果。结果　上述 17株钩体均有LipL32基因 ,但可分LipL32 1和LipL32 2两种基因型。 13个LipL32 1和 4个Li pL32 2基因型之间核苷酸和氨基酸序列同源性分别为 95 .12 %～ 96 .6 0 %和 97.79%～ 98.16 %。IPTG诱导后rMOMP1和rMOMP2表达量分别占细菌总蛋白的 4 0 %和 10 %。rMOMP1和rMOMP2均能与兔抗钩体TR patocⅠ血清发生结合反应 ,免疫家兔可对上述 17株钩体产生 1∶2～ 1∶6 4MAT效价的凝集抗体。 1∶2～ 1∶16稀释的兔抗rMOMP1和rMOMP2血清均能有效地阻断钩体的黏附。结论　所有检测的钩体具有LipL32 1或LipL32 2基因。所     

广州市海珠区2007-2010年麻疹、风疹检测结果分析

目的分析广州市海珠区2007-2010年麻疹、风疹流行特征,为麻疹、风疹的预防和控制提供可靠依据。方法对辖区内2007-2010年的麻疹、风疹病例的实验室检测结果进行统计分析。结果辖区内2007-2010年间麻疹年平均发病率为15.61/10万;风疹年平均发病率为2.57/10万。麻疹病例多集中在婴幼儿及中青年人群;风疹病例以10～25岁青少年为主,且麻疹、风疹的病例发生具有明显的季节性。结论要达到消除麻疹、控制风疹发病率的目标,应继续做好计划免疫工作,提高免疫覆盖率,严密监测,预防暴发。     

A chimeric tetravalent dengue DNA vaccine elicits neutralizing antibody to all four virus serotypes in rhesus macaques

DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general.     

Nucleic acid vaccines

There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.     

Intranasal vaccination with an adjuvanted Norwalk virus-like particle vaccine elicits antigen-specific B memory responses in human adult volunteers

Noroviruses are the most frequent cause of acute gastroenteritis in humans of all ages. No vaccines are currently available. An intranasally delivered Norwalk (NV) virus-like particle (VLP) vaccine was recently shown to be well tolerated, immunogenic and to protect against infection in Phase 1 studies. Here, we examined B memory (B(M)) responses in volunteers who received the highest dosage levels of the NV-VLP vaccine (50 μg and 100 μg). We measured the frequency of NV-specific IgG and IgA-secreting B(M) cells in peripheral blood and the level of antibodies produced by these cells in culture. All subjects immunized with 100 μg of the NV-VLP vaccine and 90% of those who received 50 μg had significant IgA or IgG B(M) responses. The B(M) cell frequencies correlated with serum antibody levels and mucosally-primed antibody-secreting cell responses. This is the first demonstration of dose-dependent, functional B(M) responses in humans immunized intranasally with a NV-VLP vaccine.     

Effect of TLR ligands co-encapsulated with multiepitopic antigen in nanoliposomes targeted to human DCs via Fc receptor for cancer vaccines

Nanoliposomes (NLs) hold promise as new highly specific nanomedicine for anti-tumor vaccines, since they could be targeted to specific receptors on dendritic cell (DC) to induce maturation and activation and increase the anti-tumor immune response. Here we studied a NLs formulation targeted or not to FcR (the receptor for the IgG Fc fragment) for the treatment of androgen-responsive prostate cancer. Luteinizing-hormone-releasing hormone (LHRH) peptide (B- and T-cell epitopes), in tandem with a tetanus toxoid T-helper epitope (830–844 region) and several TLR (Toll-Like Receptor) ligands as adjuvants were co-encapsulated. Specific uptake in vitro of LHRH-TT liposomes targeted to the FcRs of human DCs was enhanced. DC maturation/activation, cytokine production and lymphocyte activation were consistently higher in targeted than non-targeted liposomes. Similar increase was observed as more adjuvants were administrated. Targeting to specific receptor and co-encapsulation of several TLR adjuvants are essential factors for the immune response in peptide based liposome vaccine.