日本血吸虫成虫吡喹酮用药前后差异表达序列的筛选

目的应用抑制性消减杂交（SSH）技术构建吡喹酮治疗日本血吸虫感染新西兰大白兔前后肝脏内差减cDNA文库,为筛选吡喹酮治疗过程中日本血吸虫体内药物反应分子靶标奠定基础。方法日本血吸虫感染新西兰大白兔,吡喹酮治疗者为实验组,未经吡喹酮治疗者为对照组,应用PCRSelectcDNASubtraction试剂盒进行SSH分析,分别构建正向和反向消减cDNA文库,对文库进行筛选,挑取阳性克隆测序获得差异表达EST或新EST,并进行生物信息学分析。结果从2个消减文库中共筛选到39个有效阳性克隆,其中正向文库22个,反向文库17个,分析表明这些EST编码主要是一些酶及与蛋白质合成和降解相关的蛋白质。结论成功构建日本血吸虫成虫吡喹酮治疗前后消减文库,为进一步研究吡喹酮治疗血吸虫病的分子机制奠定了基础。     

基于可溶性虫卵抗原的血吸虫抗体检测免疫传感器

目的:将免疫法特异性和电化学法灵敏性相结合,建立一种高灵敏基于可溶性虫卵抗原的电化学免疫传感器,用于血吸虫抗体的检测。方法:巯基乙胺通过吸附作用在金电极表面形成的自组装单分子层(SAM),依次滴加2.5%戊二醛和血吸虫抗原(可溶性虫卵抗原,soluble egg antigen,SEA),用以捕获抗SEA抗体(兔多抗或人多抗),然后再与辣根过氧化物酶(HRP)标记的二抗形成免疫复合物。用循环伏安法检测还原峰的电流值。结果:在底物为添加了2μl 30%的双氧水及1.0 mmol/L对苯二酚的0.1 mol/L pH7.2的PBS缓冲液中,还原峰的电流值与抗SEA兔血清稀释度在10-3与10-6范围内成正比,相关系数为0.956。与抗SEA人血清稀释比在10-1与10-4范围内成正比,相关系数为0.988。在1∶40稀释度条件下,血吸虫病人与正常人血清的抗SEA还原峰的电流值相差7.4μA。结论:该方法可以用于血吸虫抗体的初步检测。     

日本血吸虫尾蚴不同途径下感染实验动物效果的观察

目的研究更为简单易行效果好的日本血吸虫感染实验动物的方法。方法腹壁剪毛贴片法、腹壁拔毛贴片法、腹壁切口滴入法。结果血吸虫尾蚴通过腹部剪毛、腹部拔毛、腹部切口后感染家兔,获虫率分别为57.6%、59.1%、61.2%;血吸虫尾蚴通过腹部剪毛、腹部拔毛、腹部切口后感染小白鼠,获虫率分别为89.3%、94.6%、96.4%。结论家兔较脆弱,用腹壁切口滴入法易切口发炎导致死亡。虽获虫率较高,不提倡使用,使用腹壁拔毛贴片法最为可行;小白鼠生命力强,用腹壁切口滴入法感染获虫率最高,不易因切口发炎导致死亡,此法提倡使用。     

日本血吸虫与HBV合并感染对幽门螺杆菌感染的影响

目的 :探讨HBV与日本血吸虫合并感染对幽门螺杆菌 (Hp)感染的影响。 方法 :利用Hp全菌抗原检测HBV、日本血吸虫单独或合并感染者血清抗HpIgG抗体。 结果 :HBV与血吸虫合并感染时 ,血清HpIgG抗体检出率 ( 85.7% )明显高于HBV或血吸虫单独感染 (阳性率分别为 4 5.5%和 72 .9% ) ,合并感染时血清抗体水平 ( 0 .87± 0 .1 0 )亦高于单独感染 ( 0 .77± 0 .2 0 ,0 .70± 0 .1 7) ,差异有显著性意义。HBV阳性者与HBV阴性者之间Hp 感染率和抗体水平相近。结论 :HBV和血吸虫合并感染对Hp感染具有协同作用     

紫外线致弱尾蚴免疫兔血清筛选血吸虫成虫cDNA文库

目的 :寻找照射致弱尾蚴中起免疫保护作用的抗原分子 ,为血吸虫病疫苗研究提供新的候选抗原。方法 :用紫外线照射致弱尾蚴免疫兔血清筛选日本血吸虫 (Schistosomajaponicum ,Sj)成虫cDNA文库 ,并对阳性克隆的插入基因片段进行PCR扩增及测序分析。结果 :经三轮筛选 ,获 10个阳性克隆 ,其插入的SjcDNA片段大小为 1 5～1.8kb。初步测序获 5个部分序列 ,其中 2个序列分别与Sj动力蛋白轻链 5 (DLC 5 )及Sj线粒体基因明显同源 ,其余 3个序列为未知的新基因片段。结论 :获得的阳性克隆插入基因片段可能为编码抗日本血吸虫感染的抗原基因     

日本血吸虫细菌人工染色体文库的初步构建(英文)

初步构建了一个日本血吸虫细菌人工染色体（BAC）文库,该文库已包含1000多个含基因组DNA插入片段的重组pEcBAC1克隆,平均插入片段为120kb。BAC文库将为日本血吸虫基因的结构与功能研究提供新的资源。     

重组Calpain蛋白的免疫原性及其诊断上的应用研究

目的研究在大肠杆菌中表达的日本血吸虫Calpain蛋白的免疫原性及其在日本血吸虫诊断上的应用. 方法用重组Calpain蛋白免疫BALB/c小鼠,ELISA法测定特异性IgG抗体的动态变化及其IgG亚型(IgG1, IgG2a, IgG2b, IgG3)产生特征;同时用重组Calpain蛋白作为诊断抗原,粪检血吸虫病阳性患者血清作为一抗,肝吸虫阳性患者血清作为考核交叉反应血清. 结果重组Calpain抗原免疫小鼠后,产生了一个极高的抗Calpain特异性抗体,免疫4周后IgG类抗体达到高峰,与对照组鼠相比较,免疫鼠血清中IgG1, IgG2a, IgG2b特异性抗体也有显著性的上升; Calpain重组抗原血清诊断日本血吸虫和粪检的符合率为100%, 粪检阳性EPG低的个体抗Calpain抗体滴度高, EPG高的患者抗Calpain抗体滴度反而低, 与肝吸虫的交叉反应率为37%. 结论日本血吸虫Calpain是一个具有免疫原性的蛋白,能够激发宿主产生高水平的免疫球蛋白,能够敏感地测定日本血吸虫的感染, 说明Calpain可以发展成为日本血吸虫病的诊断抗原和日本血吸虫疫苗候选分子.     

Schistosoma mansoni ex vivo lung-stage larvae excretory–secretory antigens as vaccine candidates against schistosomiasis

Schistosoma mansoni lung-stage larvae are known to be the major target of innate and acquired immunity to schistosomiasis. Lung schistosomula cytosolic or surface membrane antigens are hidden, entirely inaccessible to the host immune system, and hence are not particularly important as vaccine candidates. Conversely, excretory–secretory (E–S) products released from intact, viable, elongated, and contractile schistosomula are ideal potential vaccines, as such molecules can readily play a central role in the induction of local primary and memory immune response effectors that would directly target, surround, and pursue the larvae while negotiating the lung capillaries. Therefore, 6-day-old ex vivo larvae were isolated from mouse or hamster lung cells and used for generation of E–S products, which were shown to elicit strong immune responses and significant (P < 0.05) protection against challenge infection in BALB/c mice. Proteomic analysis of E–S molecules following 10× concentration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified peptides related to innumerable host and about 15 S. mansoni-specific proteins. Selected S. mansoni-specific E–S peptides prepared in a multiple antigen peptide (MAP) or recombinant form were shown to stimulate considerable specific antibody response and peripheral blood mononuclear cell expression of mRNA for several cytokines in immunized C57BL/6 and BALB/c mice. However, highly significant (P < 0.05 to <0.005) reduction in challenge infection worm burden and egg load was recorded only when the immunization conditions in test mice provided the S. mansoni antigen-specific T helper (Th) type response milieu favorable for each immunogen. That was polarized Th1 for S. mansoni aldolase and thioredoxin peroxidase 1 MAPs, polarized Th2 for recombinant 14-3-3-like protein, mixed Th1/Th17 for calpain MAP, and mixed Th1/Th2 for recombinant p18 protein. The findings together indicated that the immune responses issue is as critical as the nature and source of the antigen for the development of vaccine against schistosomiasis.     

Protective and antifecundity effects of Sm-p80-based DNA vaccine formulation against Schistosoma mansoni in a nonhuman primate model

Schistosomiasis is an important parasitic disease for which there is no available vaccine. We have focused on a functionally important antigen of Schistosoma mansoni, Sm-p80, as a vaccine candidate because of its consistent immunogenicity, protective potential and antifecundity effect observed in murine models; and for its pivotal role in the immune evasion process. In the present study we report that an Sm-p80-based DNA vaccine formulation confers 38% reduction in worm burden in a nonhuman primate model, the baboon (Papio anubis). Animals immunized with Sm-p80–pcDNA3 exhibited a decrease in egg production by 32%. Sm-p80 DNA elicited specific immune responses that include IgG; its subtypes IgG1 and IgG2; and IgM in vaccinated animals. Peripheral blood mononuclear cells (PBMCs) from immunized animals when stimulated in vitro with Sm-p80 produced appreciably more Th1 response enhancing cytokines (IL-2, IFN-γ) than Th2 response enhancing cytokines (IL-4, IL-10). PBMCs produced appreciably more spot-forming units for INF-γ than for IL-4 in enzyme-linked immunosorbent spot (ELISPOT) assays. Overall it appears that even though a mixed (Th1/Th2) type of humoral antibody response was generated following immunization with Sm-p80; the dominant protective immune response is Th1 type. These data reinforce the potential of Sm-p80 as an excellent vaccine candidate for schistosomiasis.