免疫PCR技术的策略

免疫PCR是一种新兴的免疫标记技术，具有高敏感性和特异性的特点．本文对免疫PCR的方法、DNA标记探针的构建及扩增产物的检测进行了综述．     

CpG序列的免疫激活作用研究进展(综述)

CpG序列又名免疫刺激DNA序列(ISS)，可激活多种免疫效应细胞，且无免疫原性，不会引起自身免疫疾病，具有很高的实用价值，在疾病的防治研究中有着广阔的应用前景．本文就CpG序列免疫激活作用的发现、免疫激活机理，以及该序列在疫苗和疾病治疗中的应用作一综述．     

BOG初次免疫联合结核分枝杆菌Ag85A DNA疫苗加强免疫效果的研究

为探讨BCG初次免疫,结核分枝杆菌Ag85A DNA疫苗加强免疫的序贯免疫策略对小鼠的免疫效果。采用BCG及结核分枝杆菌Ag85A DNA疫苗依次免疫小鼠,在末次免疫后的4、6、8周通过检测小鼠血清总IgG抗体、特异性淋巴细胞增殖．细胞因子的水平,观测BCG初次免疫,Ag85A DNA疫苗加强免疫的策略对小鼠的免疫效果。结果显示,采用BCG初次免疫,结核分枝杆菌Ag85A DNA疫苗加强免疫的策略组的小鼠与其它免疫方式组相比,IgG明显升高（P〈0．05）、特异性淋巴细胞明显增殖、IFN7、IL-2、IL-4水平明显增高（P〈0．05）。由此可知,在采用BCG初次免疫,结核分枝杆菌Ag85A DNA疫苗加强的免疫策略后,能增强对小鼠的免疫效应,尤其是Thl型细胞免疫反应增强明显,为进一步在动物体内进行保护性效应试验的研究提供了实验依据。     

DNA疫苗及其在卵黄抗体制备中的应用

DNA疫苗技术由于其独特的作用和优势,现已成为最有发展潜力的新兴免疫技术之一。将DNA疫苗技术和卵黄抗体技术联合起来,可获得效价更高、亲和性更好和成本更低的卵黄抗体。就DNA疫苗和卵黄抗体的各自特点以及DNA疫苗技术在卵黄抗体制备中的应用优势进行综述。     

微小隐孢子虫表面抗原CP23DNA疫苗免疫山羊诱导免疫应答及保护性研究

观察微小隐孢子虫（Cryptosporidium parvum)子孢子表面抗原CP 23 DNA疫苗免疫山羊诱导其产生免疫应答和保护性作用。将重组质粒pCR3.1-23经鼻粘免疫怀孕山羊，用ELISA测定IgG和IgA抗体滴度，用C.parvum卵囊经口对其后代攻虫感染。抗CP23抗体存在于免疫山羊的血浆和初乳中，且抗体滴度随免疫时间延长而增高。C.parvum卵囊经口感染后代，试验组山羊后代与对照组相比，卵囊排出数量减少，排出时间缩短，微小隐孢子虫表面蛋白CP 23 DNA疫苗能诱导山羊产生特异性免疫应答并对其后代有一定免疫保护作用。     

壳聚糖-卵透明带DNA纳米微囊的制备及其体外表达

目的：发展壳聚糖-卵透明带口服DNA粘膜免疫避孕疫苗。方法：首先利用基因工程技术构建含卵透明带特异抗原pZP3α DNA的真核质粒表达载体pVAX1-pZP3α，然后用壳聚糖C390包被pVAX1-pZP3α重组质粒制备免疫微囊，测定免疫微囊对质粒DNA的包裹率，用原子力显微镜(AFM)观察壳聚糖-DNA微囊的形态结构，并体外转染HeLa细胞，用间接免疫荧光法(IIF)检测pZP3α的瞬时表达。结果：pZP30αDNA正确插入到载体pVAX1中并与壳聚糖C390形成纳米免疫微囊，对重组质粒pVAX1-pZP3a的包裹率达90％以上，AFM成像技术显示这些微囊的直径为100nm到300nm不等，平均为224.6nm，形状为球形、椭圆形等。体外转染HeLa细胞用IIF法检测到了抗原pZP3α在细胞内的表达。结论：壳聚糖-卵透明带口服DNA纳米免疫微囊制备成功，为进一步发展更安全、方便、有效的卵透明带DNA避孕疫苗打下了基础。     

拉米夫定联合免疫汤治疗慢性乙型肝炎疗效分析

目的比较拉米夫定联合免疫汤和单用拉米夫定治疗慢性乙型肝炎的疗效。方法选择慢性乙肝患者84例,随机分为拉米夫定联合免疫汤组与单用拉米夫定组,每组42例,每组口服用药6个月即停药,停药后继续观察6个月。以谷丙转氨酶（ALT）、总胆红素（SB）、乙肝病毒标志物（HBsAg,HBeAg,HBV—DNA）为观察指标,比较不同治疗方案对指标的影响。结果拉米夫定联合免疫汤组与单用拉米夫定组比较,能有效地提高HBsAg、HBeAg及HBV—DNA阴转率（P〈0．05）。结论拉米夫定联合免疫汤治疗慢性乙肝疗效相对于单用拉米夫定治疗慢性乙肝疗效有更多的临床价值。     

结核分支杆菌Ag85B DNA疫苗构建、免疫原性及其保护效力

构建结核分支杆菌分泌蛋白Ag85B编码基因的DNA疫苗,研究其免疫原性和保护效力.构建该DNA疫苗并经鉴定后,应用Qiagen试剂盒大量纯化无内毒素的质粒DNA.将C57BL/6小鼠随机分为4组,分别3周间隔3次用生理盐水（A）、载体质粒（B）、卡介苗（C）、Ag85B DNA疫苗（D）免疫小鼠,通过ELISA方法检测血清中抗Ag85B抗体及其亚型.第三次免疫后28 d用结核分支杆菌H37Rv攻击,观察小鼠体重变化,攻击4周后检测细胞因子IFN-γ、IL-12,进行肺组织病理检查及菌落计数等.经Ag85B DNA疫苗免疫的小鼠特异性抗体IgG均显著高于对照组,而且IgG2b均高于IgG1和IgG2a.结核分支杆菌攻击后各组小鼠体重变化呈现不同趋势,A、B组小鼠体重从第3周开始下降;C组体重总体呈上升趋势;D组体重第2周开始下降,之后维持不变.MTT法检测脾淋巴细胞增殖实验中D组的刺激指数显著高于其它组;D组小鼠脾淋巴细胞转化率也显著高于A、B组.肺组织菌落计数C组最低,其次是D组.大体病理结果显示C组和D组肺组织病变较A、B组轻.A、B组肺泡腔内有较多渗出液,肺泡壁充血、水肿.C组肉芽肿数目最多.上述结果表明Ag85B DNA疫苗具有较强的免疫原性,呈TH1型细胞介导的免疫应答.Ag85B DNA疫苗的免疫保护效力不如BCG,有待进一步研究其应用价值.     

禽类真菌感染及其毒素中毒后免疫反应研究进展

综述了禽类抗真菌感染中体液免疫、细胞免疫及细胞因子的作用.真菌毒素通过抑制免疫球蛋白合成、效应细胞活力及抑制DNA和蛋白质合成来影响免疫机能,且免疫抑制作用与真菌毒素种类和剂量有关.最后探讨了血清学诊断技术的应用和疫苗的研发前景.     

无标度网络的免疫策略研究

文章结合目标免疫和熟人免疫各自的优势,提出了一种改进的免疫算法:随机选择网络中的部分节点,对被选节点的最大度邻居进行免疫.在算法上结合了熟人免疫的优点,即无需获知全局网络结构信息,并具有目标免疫策略的高效性.通过对算法进行基于无标度网络拓扑进行仿真模拟,验证了本算法具有更高的免疫效率和更广的应用价值.     

HBs和HCVc基因双顺反子质粒在BALB/c小鼠体内的分布

 采用注射含双顺反子质粒后,PCR法扩增不同组织中HBsAg和HCVc基因,同时检测HBV和HCV抗体应答水平.研究注射基因免疫用双顺反子质粒在小鼠组织中的分布.pcDNA3.0BApc154S₂S 1次注射BALB/c小鼠后,24 h内主要分布于血液、肝、脾、骨髓、淋巴结、注射部位肌肉、肺和肾等含血液丰富的组织中.注射后2 d主要在骨髓、血液和注射部位肌肉中存在.7 d后仅在注射部位肌肉中能检测出来,并可持续到第9周.组织切片镜检质粒注射初期肌肉细胞轻度浊肿,随后肌膜细胞轻度增生,未见其它明显的组织病理学变化.质粒多次免疫BALB/c小鼠未见明显的临床症状和病理组织学变化.质粒在小鼠体内不同组织存在时间不一致.     

Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon-alpha/beta and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3' non-translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP-RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.     

未来的丙型肝炎疫苗

丙型肝炎病毒（hepatitis C virus,HCV）是丙型肝炎的病原体,是导致输血后肝炎和非甲非乙型肝炎的主要致病因子。HCV感染常导致慢性肝炎、肝硬化和肝细胞癌。HCV在细胞内和动物体内复制能力很差,没有理想的细胞模型和动物模型。丙型肝炎目前尚无疫苗,还有很多病人在等待治疗。研制预防性和治疗性疫苗,以控制HCV感染有着重要意义。经过几代科技工作者的努力和奉献,丙型肝炎疫苗研究取得了重大进展。用核酸疫苗作首次免疫,用多肽疫苗或树突状细胞疫苗作加强免疫,不但有预防作用,而且有治疗效果。核酸疫苗将为改善人类和动物健康作出巨大贡献。     

分支DNA探针法检测21例丙型肝炎血清病毒RNA

目前主要依赖检测丙型肝炎抗体来确定对丙型肝炎病毒(Hepatitis C virus,HCV)感染的诊断,但它不能反应机体是否有活动的病毒血症。分支DNA探针法应用合成的DNA分子与靶HCV—RNA特异性的杂交,形成RNA—DNA杂交体,用dioxetane作为底物与化学发光物结合,通过测定其发光强度可直接检测血清HCV—RNA的含量。本文测定21例慢性活动性丙型肝炎血清。HCV—RNA最低值为0.66Meg/ml;最高值为58Meg/ml,21例中86％的数值分布在0.66Meg/m-12Meg/ml的范围内。此方法cutoff值为0.5Meg//ml。我们所测定的21例均高于cutoff值。此方法操作简便,特异性强。为临床丙型肝炎治疗的监测及疗效的判断提供了重要的依据。     

丙型肝炎病毒核酸疫苗的研究

从丙型肝炎病人的阳性血清中提取出丙型肝炎病毒ＲＮＡ，通过ＲＴ－ＰＣＲ的方法获得丙型肝炎病毒Ｃ区基因片段。并将此片段克隆到真核细胞表达载体ｐｃＤＮＡ３．１（＋），得到重组质粒ｐｃＤＮＡ－ＨＣＶ／Ｃ，再将其通过肌肉注射免疫ＢＡＬＢ／小鼠后，小鼠产生了抗ＨＣＶ／Ｃ区抗体。     

Evasion of intracellular host defence by hepatitis C virus

Viral infection of mammalian cells rapidly triggers intracellular signalling events leading to interferon alpha/beta production and a cellular antiviral state. This 'host response' is our first line of immune defence against infection as it imposes several barriers to viral replication and spread. Hepatitis C virus (HCV) evades the host response through a complex combination of processes that include signalling interference, effector modulation and continual viral genetic variation. These evasion strategies support persistent infection and the spread of HCV. Defining the molecular mechanisms by which HCV regulates the host response is of crucial importance and may reveal targets for novel therapeutic strategies.     

Adaptive immune responses in acute and chronic hepatitis C virus infection

The hepatitis C virus (HCV) persists in the majority of infected individuals and is a significant cause of human illness and death globally. Recent studies have yielded important insights into immunity to HCV, in particular revealing the central role of T cells in viral control and clearance. Other key features of adaptive immune responses remain obscure, including mechanisms by which T cells control HCV replication, the role of antibodies in conferring protection and how cellular and humoral immunity are subverted in persistent infection.     

HCV感染与多种自身抗体高检出率的关系

为探讨丙型肝炎病毒（ＨＣＶ）感染后体内出现的自身抗体的病理意义及其引起的自身免疫的发生机制。方法：对１６１例ＨＣＶ感染者进行了八种自身抗体（抗核抗体、类风湿因子、抗甲状腺球蛋白抗体、抗甲状腺微粒体抗体、抗双链ＤＮＡ抗体、抗ＲＮＰ抗体、抗Ｓｍ抗体、抗精子抗体）的检测。结果：有５２例检出６９项次自身抗体,自身抗体检出率为３２．３％,显著高于健康人对照组（Ｐ＜０．００５）（未计抗精子抗体）。结论：ＨＣＶ感染可能是诱发自身免疫反应的一个重要因素     

Interferon modulation of cellular microRNAs as an antiviral mechanism

RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.     

Specific immune responses induced by a multi-epitope antigen of hepatitis C virus in mice and rabbits

Five highly conserved and immunogenic epitopes of hepatitis C virus (HCV) have been chosen to form a multi-epitope antigen gene and fused with β-galactosidase gene to express a hybrid GZ-PCX antigen, which could be specifically recognized by human HCV sera. High level of anti-GZ-PCX IgG has been induced when mice or rabbits were immunized with GZ-PCX antigen emulsificated with complete Freund’s adjuvant or mixed with killed attenuatedSalmonella typhimurium SL3261. The specific anti-GZ-PCX IgG reached a high titer of 10^-6, which remained for several months. Specific cytotoxic T lymphocyte (CTL) effects, delayed type hypersensitivity reaction (DTH) and proliferation of peripheral lymphocytes have been induced by GZ-PCX antigen or synthetic peptides. High level of anti-GZ-PCX slgG has been detected in mice’s intestinal washing fluids, which indicates that the antigen induced mucosal immunity as well as systematic immunity. The studies show that the HCV multi-epitope antigen induces high level of specific immune responses without obvious toxicity, which might be able to provide protectivity to any HCV genotypes and isolates.