心脏基因工程研究对一级康复的干预作用：构建柯萨奇B2病毒毒粒蛋白候选基因疫苗诱导体液免疫的评估

目的：探讨心脏基因工程研究中构建柯萨奇B2病毒毒粒蛋白侯选基因疫苗，并评价其诱导体液免疫的效果：方法：实验于2003—05／12在中国农业科学院国家生物重点实验室哈尔滨兽医研究所生物二室完成。采用反转录聚合酶链反应技术扩增柯萨奇B2病毒的主要中和抗原毒粒蛋白基因，通过分子克隆构建pcDNA3-柯萨奇B2病毒毒粒蛋白基因免疫质粒。选择3周龄BALB／c雄性小鼠30只，随机分成3组，pcDNA3-柯萨奇B2病毒毒粒蛋白组：对小鼠进行pcDNA3-柯萨奇B2病毒毒粒蛋白免疫；空载体对照组对小鼠进行pcDNA3免疫；磷酸盐缓冲液组：对小鼠进行磷酸盐缓冲液免疫，每组10只。每组免疫时间和接种量、接种方法相同。采用酶联免疫吸附法测定pcDNA3．柯萨奇B2病毒毒粒蛋白、pcDNA3、磷酸盐缓冲液免疫小鼠后2，4，6，8周柯萨奇B2病毒特异性抗体免疫球蛋白C水平。结果：①获得pcDNA3-柯萨奇B2病毒毒粒蛋白基因免疫质粒，可在HeLa细胞中表达。②免疫后6，8周pcDNA3-柯萨奇B2病毒毒粒蛋白组柯萨奇B2病毒特异性抗体免疫球蛋白G水平明显高于免疫后4周似4靠0．153&;#177;0．013，0．150&;#177;0．011，0．142&;#177;0．014，P〈0．01，0．05）；免疫后4周pcDNA3-柯萨奇B2病毒毒粒蛋白组柯萨奇B2病毒特异性抗体免疫球蛋白G水平明显高于空载体对照组和磷酸盐缓冲液组（0．120&;#177;0．007，0．119&;#177;0．009．P〈0.011。结论：pcDNA3-柯萨奇B2病毒毒粒蛋白可诱导小鼠产生体液免疫，做为候选基因疫苗，以基因免疫的角度参与心脏基因工程研究．有望为该病毒性心肌炎的发生起到一级预防作用。     

心脏基因工程研究：柯萨奇B2病毒云南分离株VP1基因的分子特征

目的：探讨CVB2云南分离株编码主要中和抗原的衣壳蛋白VP1基因?B5姆肿犹卣鳎云谖泄鶦VB2感染致病的分子基础、基因疫苗的研制和心脏基因工程实施提供参考依据。方法：采用逆转录PCR技术扩增CVB2云南分离株VP1基因，经分子克隆获得pMD18-T-CVB2VP1并测序，应用生物信息学进行其序列、同源性分析、预测蛋白质二级结构，并建立进化树。结果：CVB2云南分离株VP1基因全长约846bp，无起始密码及终止密码，与Ohio-1株(GenBank登录号：AF081312)核苷酸、氨基酸序列同源性最高为98％。BC环的BB区与BC区之间缺失6个腺嘌呤，其编码2个构成无规卷曲结构的赖氨酸。CVB的共同抗原表位区中RIYFKPKHVKA高度保守，为云南分离株及其他参考毒株的共同序列，变异主要在CVB2云南株的251～252位，W→Y，V→I。结论：CVB2云南分离株与Ohio-1株同源性最高，亲缘关系最近，以主要中和抗原表位区存在缬氨酸缺失，CVB共同抗原表位区存在具有规律性的变异及保守序列等为其主要分子特征。     

心脏基因工程研究:柯萨奇B2病毒云南分离株VP1基因的分子特征

目的:探讨CVB2云南分离株编码主要中和抗原的衣壳蛋白VP1基因的分子特征,以期为中国CVB2感染致病的分子基础、基因疫苗的研制和心脏基因工程实施提供参考依据。方法:采用逆转录PCR技术扩增CVB2云南分离株VP1基因,经分子克隆获得pMD18-T-CVB2VP1并测序,应用生物信息学进行其序列、同源性分析、预测蛋白质二级结构,并建立进化树。结果:CVB2云南分离株VP1基因全长约846bp,无起始密码及终止密码,与Ohio-1株(GenBank登录号:AF081312)核苷酸、氨基酸序列同源性最高为98%。BC环的βB区与βC区之间缺失6个腺嘌呤,其编码2个构成无规卷曲结构的赖氨酸。CVB的共同抗原表位区中RIYFKP-KHVKA高度保守,为云南分离株及其他参考毒株的共同序列,变异主要在CVB2云南株的251～252位,W→Y,V→I。结论:CVB2云南分离株与Ohio-1株同源性最高,亲缘关系最近,以主要中和抗原表位区存在缬氨酸缺失,CVB共同抗原表位区存在具有规律性的变异及保守序列等为其主要分子特征。     

柯萨奇病毒B4 2B基因siRNA达载体的构建及其对柯萨奇病毒B4抑制作用的研究

目的构建针对柯萨奇病毒B4 2B基因的siRNA表达载体并检测该载体在体外培养细胞中对柯萨奇病毒B4的抑制作用.方法选择柯萨奇病毒B4的2B基因区21bp的基因片段作为靶序列,合成含有靶序列的DNA片段并克隆到pGCsi-U6/Neo/GFP/siNeGative载体中,构建pGCsi-U6/Neo/GFP/2B,使其可表达具有发夹结构的双链siR-NA,在转染该载体后用柯萨奇病毒B4攻击Hela细胞,检测该载体对病毒感染细胞的保护效应.结果通过酶切、电泳、序列分析及荧光显微镜观察证明载体构建成功,而且pGCsi-U6/Neo/GFP/2B转染组的柯萨奇病毒B4的滴度及TCID50都明显低于pGCsi-U6/Neo/GFP/siNeGative转染组.结论成功构建了柯萨奇病毒2B基因的siRNA表达载体,而且该载体在体外培养细胞中对柯萨奇病毒的复制具有抑制作用.     

柯萨奇病毒B4 2B基因siRNA表达载体的构建及其对柯萨奇病毒B4抑制作用的研究

目的构建针对柯萨奇病毒B42B基因的siRNA表达载体并检测该载体在体外培养细胞中对柯萨奇病毒B4的抑制作用。方法选择柯萨奇病毒B4的2B基因区21bp的基因片段作为靶序列，合成含有靶序列的DNA片段并克隆到pGCai-U6／Neo／GFP／siNeGative载体中，构建pGCsi-U6／Neo／GFP／2B，使其可表达具有发夹结构的双链siRNA，在转染该载体后用柯萨奇病毒B4攻击Hela细胞，检测该载体对病毒感染细胞的保护效应。结果通过酶切、电泳、序列分析及荧光显微镜观察证明载体构建成功，而且DGCsi-U6／Neo／GFP／2B转染组的柯萨奇病毒B4的滴度及TCID50都明显低于pGCsi-U6／Neo／GFP／siNeGative转染组。结论成功构建了柯萨奇病毒2B基因的siRNA表达载体，而且该载体在体外培养细胞中对柯萨奇病毒的复制具有抑制作用。     

柯萨奇B组病毒与小儿急性短暂性跛行发病关系的研究

目的探讨小儿下肢急性短暂性跛行发病的感染因素,为临床提供诊断依据。方法采用酶联免疫吸附试验对600例小儿急性跛行患儿组、100例上呼吸道感染组(下称上感组)、100例健康儿童对照组进行血清柯萨奇B组病毒1～6型(CVB 1～6型)IgM检测。结果小儿跛行患者组CVB-IgM阳性检出率为51.3%,其中以B1型为主,B4型次之,阳性率分别占36.8%和16.3%,检出双重阳性感染79例,占总阳性病例的25.7%(79/308);上感组CVB-IgM检出率为29.0%,型别亦以B1和B4为主,检出率依次为15.0%和10.0%,双重阳性占阳性病例的20.7%(6/29);健康对照组CVB-IgM检出率为16.0%,型别仍以B1和B4为主,检出率分别为6.0%和5.0%。3组的阳性率跛行组明显高于上感组和健康对照组,差异均有统计学意义(P<0.01)。结论柯萨奇B组病毒感染与小儿急性短暂性跛行发病关系密切,可能是其发病的重要感染因素之一,分型检测CVB特异性IgM抗体具有重要的临床诊断意义。     

不同免疫类型的柯萨奇病毒DNA疫苗免疫效果研究

柯萨奇病毒B组3型（CoxsackievimsgroupBtype3，CVB3）是引起人类病毒性心肌炎的主要病原体，目前还没有特异性的防治措施。我们将巨噬细胞源趋化因子（macrophage—derivedchemokine，MIC）基因和志贺毒素（Shigatoxin，STx）B亚单位基因分别与CVB3VP1基因融合构建DNA疫苗pcDNA3／MDC-VPI和pcDNA3／SYxB—VPI，免疫小鼠，用7LD30CVB3致死攻击，比较两种疫苗的免疫效果，为柯萨奇病毒疫苗的研制奠定试验基础。     

柯萨奇病毒B5基因组进化存在结构蛋白编码区VP4的重组

目的重组是肠道病毒进化的重要机制,大多数重组事件发生在包括P2和P3的肠道病毒的非编码区中,但是在埃可病毒30株中首次发现了作为蛋白质编码区的VP4区域中的重组,本研究旨在探讨柯萨奇病毒B5(CVB5)中是否存在类似的重组事件。方法从GenBank中检索CVB5的核苷酸序列,分别在5′UTR和VP1区域中进行系统发育分析。此外,跨越5′UTR-VP4-5′VP2的部分基因组用于检测重组并使用序列相似性作图(Similarity plot)来分析重组体。结果 5′UTR和VP1系统发育树显示5′UTR和VP1之间有4个病毒株位于不同簇中,表明这些病毒株中存在重组事件。Similarity plot分析表明,CVB5AY875692的5′UTR-VP4-5′VP2区域存在交叉,序列分析证明VP4区域存在重组位点。结论 CVB5在VP4区域具有重组事件,表明结构蛋白编码区亦可作为CVB5重组的候选基因位点。     

柯萨奇病毒B诱导的免疫性多发性肌炎模型的建立

目的：探讨制作实验性免疫性多发性肌炎动物模型的方法，以及柯萨奇病毒感染与多发性肌炎发病的关系。方法：分别用不同毒力0．3ml的柯萨奇病毒B1（CVB1，10^-2，10^-3,10^-4 TCID50）感染和兔肌肉匀浆（30mg/ml）加完全弗氏佐剂多次免疫28只正常豚鼠，共观察3～5周；和不同毒力0．2mlCVB1，10^-2，10^-3,10^-4 TCID50分别腹腔接种各20只BALB／C和C57BL／6小鼠；以及0．1ml CVB3 10^-6 TCID50腹腔接种28只BALB／C小鼠，共观察1～3周，制成实验性多发性肌炎动物模型，观察其在血清肌酶、巨检和组织病理的改变。结果：发现0．3训毒力为10^-2（A组），10^-3（B组）和10^-4（C组）TCID50柯萨奇病毒B感染豚鼠纽，3周后仅少数豚鼠出现多发性肌炎的症状，但均能导致肌酶谱（CK，CK-MM，LDH和AST）异常升高，与正常组相比有明显差异（均P〈0．05），以B组升高值最高，但巨检肌肉无异常；4周后10^-3组（F组）豚鼠33．3％出现下肢瘫痪，巨检发现肌肉萎缩，弹性差，病理检查证实为多发性肌炎改变；3周与5周的CK和CK-MM升高值相比，无显著差异（P=0．1031和P=0．1164）。单纯兔肌肉匀浆免疫（E组）也能导致豚鼠肌酶谱异常升高，但不如病毒感染后明显，也不引起股肌肉肌炎的病理改变。柯萨奇病毒B1和B3均不能诱导BALB／C和C57BL／6小鼠出现多发性肌炎改变。结论：柯萨奇病毒B1感染的毒力和病毒量与豚鼠多发性肌炎的发病相关，在感染5周后比较明显。本模型可作为研究人类多发性肌炎的一个重要手段，为人类肌炎的发病机理和治疗提供较好的动物模型。     

商陆提取蛋白体外抗柯萨奇病毒的实验研究

目的:探讨商陆提取蛋白(PAP)在体外抗柯萨奇病毒(CVB3)的效应。方法:(1)用MTT法检测PAP对Hela细胞的毒性作用;(2)在Hela细胞上采用微量细胞病变抑制法观察PAP直接灭活CBV3的作用。结果:(1)连续作用6dPAP对Hela细胞的50%毒性浓度依次降低;(2)实验的第1、2、3d,0.1至25mg/L-3浓度的PAP能抑制100TCID50和1000TCID50柯萨奇病毒在胞内复制;(3)实验的第4、5、6d,0.1至25mg/L-3浓度的PAP作用于感染了CVB3的Hela细胞发生了病变。结论:在体外细胞培养上,商陆提取蛋白能起到阻断或减缓柯萨奇病毒吸附细胞的作用。     

Intravascular naked DNA vaccine encoding glycoprotein B induces protective humoral and cellular immunity against herpes simplex virus type 1 infection in mice

Naked plasmid DNA (pDNA) vaccine expressing herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) was tested for protective activity against acute HSV-1 infection in mice. The pDNA was intravenously injected into Balb/c mice via their tail vein under high pressure, and the vaccination was performed two times at an interval of 7 days. The gB gene vaccination significantly protected the mice from subsequent intraperitoneal challenge with a lethal dose of HSV-1, which killed all the animals given control plasmid or saline. The protective activity was correlated with the dose of the plasmid inoculated, the survival rate reaching 83% in mice vaccinated with 5 microg of pDNA. The vaccinated mice were also protected from latent HSV infection. The immunized mice showed significant elevation in neutralizing antibody against HSV-1 as well as serum levels of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma). When mice were immunized with 5 microg of an Epstein-Barr virus (EBV)-based plasmid vector harboring the gB, the cytotoxic T lymphocytes (CTLs) activity and proliferative response for HSV-1 were also induced. The results strongly suggest that intravenous immunization of naked pDNA may induce humoral and cellular immune responses against the virus, leading to a significant prophylactic outcome against HSV-1 infection in mice.     

HSV-gD2-IL-2融合蛋白疫苗动物免疫的研究

目的:研究gD2-IL-2融合蛋白疫苗对BALB/C小鼠生殖器疱疹动物模型的保护能力.方法:gD2-IL-2融合蛋白疫苗和PBS、IL-2分别免疫60只雌性小鼠,免疫前后ELISA法观察血清抗体变化.小鼠阴道内接种HSV-2-Sav株建立小鼠生殖器疱疹模型,感染后观察小鼠阴道病变情况及病死率,同时行阴道分泌物病毒量检测.结果:疫苗组小鼠免疫后抗体滴度明显升高,与其他2组比较,差异有统计学意义(P<0.01).所有小鼠感染后均出现外阴部红肿、水疱等症状,但疫苗组阴道症状得分和病死率与其他2组相比,差异有统计学意义(P<0.01).结论:制备的HSV-gD2-IL-2融合蛋白能够在一定程度上激发免疫小鼠产生HSV-2-IgG和中和抗体,初步证实疫苗在抵抗致死性病毒攻击时对小鼠有较好地保护作用.     

Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death

Virulent Mycobacterium tuberculosis (Mtb) induces a maladaptive cytolytic death modality, necrosis, which is advantageous for the pathogen. We report that necrosis of macrophages infected with the virulent Mtb strains H37Rv and Erdmann depends on predominant LXA₄ production that is part of the antiinflammatory and inflammation-resolving action induced by Mtb. Infection of macrophages with the avirulent H37Ra triggers production of high levels of the prostanoid PGE₂, which promotes protection against mitochondrial inner membrane perturbation and necrosis. In contrast to H37Ra infection, PGE₂ production is significantly reduced in H37Rv-infected macrophages. PGE₂ acts by engaging the PGE₂ receptor EP2, which induces cyclic AMP production and protein kinase A activation. To verify a role for PGE₂ in control of bacterial growth, we show that infection of prostaglandin E synthase (PGES)^−/− macrophages in vitro with H37Rv resulted in significantly higher bacterial burden compared with wild-type macrophages. More importantly, PGES^−/− mice harbor significantly higher Mtb lung burden 5 wk after low-dose aerosol infection with virulent Mtb. These in vitro and in vivo data indicate that PGE₂ plays a critical role in inhibition of Mtb replication.     

Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria

We show here an effective and novel approach to engineer peptide-based vaccines using a chemically defined system, known as multiple peptide antigen systems (MAPs), to protect an inbred mouse strain from infection against rodent malaria. 10 mono- and di-epitope MAP models containing different arrangements and stoichiometry of functional B and/or T helper cell epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize A/J mice. While these mice did not respond to the mono-epitope MAP bearing only the B or T epitope, very high titers of antibody and protective immunity against sporozoite challenge were elicited by di-epitope MAPs, particularly those with the B and T epitopes in tandem and present in equimolar amounts. These results, obtained in a well-defined rodent malaria model, indicate that MAPs may overcome some of the difficulties in the development of synthetic vaccines, not only for malaria but also for other infectious diseases.     

Interleukin-10 prevents spontaneous death of germinal center B cells by induction of the bcl-2 protein.

In this study, we show that IL-10 enhances in vitro the viability of purified splenic B cells. There was a two- to threefold increase in recovery of viable cells during a 15-d culture period in the presence of IL-10. This effect was abolished by neutralizing antibodies to IL-10. The survival of large splenic B cells, which mostly represent follicular center cells, was similarly increased. The in vitro rescue from spontaneous death of the latter cells is known to involve a bcl-2-dependent pathway. We therefore investigated whether IL-10 might affect bcl-2 expression. Unseparated B cells as well as large splenic B cells displayed a strong expression of bcl-2 protein by immunofluorescence at days 2-7 of culture in the presence of IL-10. Other lymphokines such as IL-2 and IL-4 were able to trigger only a transient and faint expression of bcl-2; moreover, this effect was abolished by anti-IL-10 mAb. Inasmuch as activated B cells can produce their own IL-10, this lymphokine may play a crucial role in relieving from apoptosis those B cells that encounter their antigen in B cell follicles.     

Up-regulation of adhesion molecule expression and induction of TNF-alpha on vascular endothelial cells by antibody against human parvovirus B19 VP1 unique region protein

BACKGROUND: Human parvovirus B19 infection has been frequently described as a cause or trigger of various autoimmune diseases. In previous studies, we have postulated the association among human parvovirus B19 (B19)-VP1 unique region (VP1u), production of anti-beta2-glycoprotein I (anti-beta2GPI) antibody and anti-phospholipid syndrome (APS)-like autoimmunity. However, the precise role of B19-VP1u in induction of APS is still obscure. METHODS: To further elucidate the pathogenic roles of VP1u in B19 infection and autoimmunity, we examined the effect of anti-B19-VP1u IgG antibodies on endothelial cells that is recognized to play crucial roles in APS. Human vascular endothelial cells, ECV-304, were incubated with various preparations of purified human or rabbit IgG. The activation of endothelial cells and production of cytokines were assessed by flow cytometry and ELISA, respectively. RESULTS: Purified IgG from rabbits immunized with recombinant B19-VP1u proteins can up-regulate ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), MHC class II (HLA-DR, DP, DQ) molecule expression, and TNF-alpha production in endothelial cells as compared to those endothelial cells cultured with control IgG. Additionally, significantly increased phosphorylated-P38 mitogen-activated protein kinase (P38 MAPK) and iNOS were observed in both human anti-beta2GPI IgG and rabbit anti-B19-VP1u IgG treated-ECV-304 cells, respectively. CONCLUSIONS: These experimental results imply that antibodies against B19-VP1u play important roles in the immunopathological processes as well as human anti-beta2GPI IgG that leads to development of APS by involving p38 phosphorylation and iNOS activation. It could provide a clue in understanding the role of anti-B19-VP1u antibodies in APS manifestations.     

PC-1基因功能研究:PC-1蛋白兔多克隆抗体制备的改进和应用

目的:获得高效价PC-1蛋白的兔多克隆抗体,用于PC-1功能的实验。方法:实验于2004-02/08在解放军军事医学科学院生物工程研究所完成。以纯化后的PC-1蛋白及其N端46个氨基酸与GST的融合蛋白(GST-PC-1和GST-PC-1-46)为抗原,通过一种经淋巴结注射、免疫家兔的改进的免疫方法制备兔多克隆抗体。应用ProteinA亲和层析纯化抗体。以酶联免疫吸附和Western-blot及免疫组化法鉴定所得抗体。结果:①2个兔多克隆抗体的效价分别为1∶12800和1∶51200。②PC-1在高恶性前列腺癌细胞C4-2中高表达,在低恶性前列腺癌细胞LNCaP中有少量表达。③经免疫组化染色后,含有PC-1蛋白的C4-2及LNCaP细胞均呈阳性反应,细胞质中可见明显着色,而空泡状的胞核则没有着色,提示PC-1蛋白主要分布在细胞浆中。结论:用一种改进的免疫方法,获得高效价的、可应用于酶联免疫吸附和Western-blot及免疫组化实验的PC-1蛋白兔多克隆抗体。