广州管圆线虫的抗原定位研究

目的 为广州管圆线虫抗原的分离，纯化及广州管圆线虫病的免疫诊断等提供实验依据。方法 运用间接荧光抗体试验（IFAT）和免疫金银染色试验（IGSS）对广州管圆线虫的抗原定位进行研究。结果 在IFAT中，广州管圆线虫的肠管壁、子宫内虫卵的卵细胞，卵巢和皮层具有特异性荧光，提示这些器官具有抗原性，而在IGSS中，广州管圆线虫的抗原定位器官只有肠管壁，子宫内虫卵的卵细胞和卵巢，而不包括皮层。结论 虫体的肠管壁是主要的抗原定位器官，虫体子宫内虫卵和卵巢亦显示有抗原性。这些器官中的抗原与感染宿主的免疫密切相关。     

应用免疫金银染色法对日本血吸虫、华支睾吸虫和卫氏肺吸虫进行抗原定位的研究

应用免疫金银染色技术（IGSS）和间接荧光抗体试验（IFAT）对日本血吸虫、华支睾吸虫和卫氏肺吸虫进行抗原定位的研究。证明日本血吸虫成虫主要特异性抗原定位在虫体肠管壁,华支睾吸虫和卫氏肺吸虫主要抗原定位也在肠管壁。IGSS观察华支睾吸虫的贮精囊部位以及卫氏肺吸虫皮层也存在特异性抗原。     

免疫印迹法分析广州管圆线虫成虫抗原的探讨

为了解广州管圆线虫成虫虫体蛋白组分,从中寻找特异性抗原,并为广州管圆线虫分类学提供分子生物学资料,我们应用免疫印迹法对其成虫蛋白质进行分析,结果如下:一、材料与方法(一)虫体来源1.广州管圆线虫成虫:用人工消化法从褐云玛瑙螺分离出广州管圆线虫第3期幼虫,经腹腔注射感染大白鼠,待虫体完全成熟后(39d 以上)解剖大白鼠,从肺动脉取出。     

华支睾吸虫抗原定位,生化及免疫学特性初步分析

本文在制备了华支睾吸虫全虫粗抗原（ＣｓＡｇ）、代谢抗原（ＥｓＡｇ）、表膜抗原（ＭＡｇ）及其相应抗血清的基础上，应用石蜡和冰冻两种连续组织切片、ＩＦＡＴ、ＩＧＳＳ、ＰＡＰ三和免疫化学方法对华支睾吸虫抗原定位进行了较系统的研究。结果表明：虫体定位器官主要是肠管，睾丸，储精囊和虫卵卵黄膜。两种切片抗原定位的主要区别是虫体皮层，石蜡切片未见皮层有抗原性，而冰冻切片虫体皮层有抗原性性。在此基础上应用ＩＥ、Ｓ     

福寿螺体内广州管圆线虫Ⅲ期幼虫的形态学观察

目的观察福寿螺体内广州管圆线虫Ⅲ期幼虫的形态学特征。方法实验室培养的广州管圆线虫Ⅰ期幼虫感染禁食24h的福寿螺,61d解剖,取其肺囊和足肌,常规制作石蜡切片,观察其Ⅲ期幼虫外部形态和内部结构。结果Ⅲ期幼虫虫体卷曲,头部圆钝,咽管始于头部顶端的口孔,在咽肠连接处与肠管连接,尾部尖,肛管清晰。幼虫皮层为伊红染色,皮层外有一层无色透明的鞘膜。部分虫体尾部出现圆柱体,有些幼虫体内出现亚腹腺、很短的双管子宫等Ⅳ期幼虫早期特征。结论广州管圆线虫Ⅲ期幼虫的形态学特征清晰,对预防广州管圆线虫病流行有一定意义。     

四种抗原间接酶联免疫吸附试验诊断广州管圆虫病实验研究

用广州管圆线虫雌虫全虫及其消化系统、生殖系统、体壁分别制备四种粗抗原，用间接酶联免疫吸附试验（ＥＬＩＳＡ）检测广州管圆线虫感染大鼠血清、曼氏迭宫绦虫裂头蚴抗原免疫大鼠血清，猪蛔虫抗原免疫大鼠血清及日本血吸虫感染大鼠血清抗体。结果，对广州管圆线虫感染大鼠血清四种抗原，ＥＬＩＳＡ阳性率和ＯＤ比值比较，差异无显著性，均具有较高敏感性和特异性。所用四种粗抗原分别与三种异源性血清进行交叉试验，结果，除一份猪蛔虫抗原免疫大鼠血清外均为阴性，广州管圆线虫感染大鼠血清ＯＤ比值均高于三种异源性血清。     

广州管圆线虫成虫可溶性抗原诱导小鼠保护性免疫的研究

目的 探讨广州管圆线虫成虫可溶性抗原诱导小鼠的保护性免疫,为广州管圆线虫病的免疫预防研究提供科学依据。方法 以广州管圆线虫成虫可溶性抗原隔周腹腔注射免疫小鼠1次,共3次,末次免疫1w后,用300条广州管圆线虫第三期幼虫感染每鼠,攻击感染4w后解剖每鼠检获虫体,计算减虫率,并观察、测量检获虫体形态、大小。此外,还检测了小鼠体内血清特异性抗体IgG和血液嗜酸性粒细胞含量。结果 免疫组小鼠与对照组比较,有极显著的减虫效果（P＜0．01）,800μg、400μg和200μg免疫原组小鼠的减虫率分别是44．0％、42．6％和18．7％。免疫组小鼠体内检获的虫体比对照组要少。免疫组小鼠血清抗体水平及血液嗜酸性粒细胞绝对值均高于对照组（P＜0．01）。结论 广州管圆线虫成虫可溶性抗原可诱导小鼠产生保护性免疫。     

广州管圆线虫特异性基因的检测

目的广州管圆线虫特异性基因的探索和检测以及其应用诊断价值。方法根据广州管圆线虫的特异性rRNA基因的序列特点,设计引物,对广州管圆线虫成虫、Ⅲ期幼虫及Ⅰ期幼虫及虫卵的DNA进行PCR扩增,并对扩增产物进行测序。结果经电泳和DNA序列测定,广州管圆线虫各期虫体及虫卵的rRNA基因片段有特异性。结论广州管圆线虫的大亚基rRNA基因是细胞中含量最多,且结构保守,编码为多拷贝的基因,易于检测发现,具有进行诊断广州管圆线虫的价值。     

日本血吸虫感染过程抗原定位的动态变化研究

本实验用ＩＦＡ方法，用日本血吸虫感染过程中的各期兔血清进行了成虫和虫卵的抗原定位研究，以观察抗原的定位随感染进程的动态变化。结果表明，用Ｒｏｓｓｍａｎ＇ｓ固定液固定的成虫石蜡切片的抗原定位及反应强弱依次为肠管壁、间质和皮层。雄性生殖系统，如睾丸可见荧光反应。雌性生殖系统未见荧光反应。雄虫的反应强度比雌虫强，尤其在间质部位最明显。成虫冰冻切片抗原定位反应最强的部位在皮层。间质较弱，肠管壁几乎阴性。对病鼠肝内虫卵抗原定位结果显示，石蜡切片抗原从感染后第４周起可见定位在虫卵内毛蚴的顶腺、侧腺及卵黄膜、卵间隙和卵壳上。顶腺和侧腺的阳性反应最强。第五周开始可见卵周有何博礼现象。冰冻切片的抗原也在第四周开始出现，定位在卵黄膜及卵壳上。     

不同发育阶段广州管圆线虫的抗原分析

目的 分析不同发育阶段广州管圆线虫抗原差异 ,筛选优势诊断抗原分子。　方法　对不同发育阶段的广州管圆线虫的虫体蛋白进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和免疫印迹分析。结果　不同发育阶段的虫体蛋白质谱大致相同 ,SDS PAGE中出现的少数差异带在免疫印迹试验(Westernblotting)中无明显差异。各期虫体抗原相对分子质量为Mr 40 000、50 000、 66 000和 80 000的 ,与感染大鼠和正常大鼠血清均出现反应条带。 2～3周幼虫Mr 104 000与 2周感染血清出现明显反应条带。雌虫Mr 33 000及所有虫体的Mr 32 000抗原与感染后 2周血清出现反应条带 ,与感染 3周～5月的大鼠血清均出现明显反应 ,与正常大鼠血清均无明显反应。　结论　Mr 40 000、50 000、66 000和80 000抗原可能在以虫体粗抗原作探针的免疫诊断中引起非特异性反应。幼虫Mr 104 000、雌虫Mr 33 000和所有虫体的Mr 32 000可作为广州管圆线虫病早期诊断和流行病学调查的候选抗原分子。     

广州管圆线虫在长爪沙鼠体内的发育及其引起的病理损害

目的观察广州管圆线虫在长爪沙鼠体内的发育及其引起的病理损害。方法以广州管圆线虫第Ⅲ期幼虫经腹腔注射感染成年长爪沙鼠（10条幼虫／鼠）,感染后不同时间检查脑、心肺组织内的虫体,肉眼观察和病理切片检查脑、肺组织,于感染后第46d采用直接涂片法检查粪便。结果从感染的长爪沙鼠脑和心肺组织共检获虫体127条（♀62／♂65）,平均虫数为（3．63±1．46）／鼠,感染后第23－30d,检获虫体数最多;虫体在感染后30d内主要分布在脑,30d后主要分布在肺;幸存的长爪沙鼠大多数广州管圆线虫能在其肺中发育至成虫并产卵,同时在部分沙鼠粪便中检查到Ⅰ期幼虫;病理结果显示蛛网膜下腔可见虫体,肺内有虫卵,广州管圆线虫从脑移行至肺导致嗜酸性粒细胞增多性脑膜炎和肉芽肿性肺炎。结论广州管圆线虫可在长爪沙鼠体内发育成熟并造成脑和肺组织的损害。     

日本血吸虫虫卵中毛蚴抗原的融合表达及抗原性分析

目的 重组表达制备日本血吸虫虫卵中毛蚴抗原（SjMP10）。 方法 根据日本血吸虫虫卵中毛蚴抗原SjMP10分子的读框序列设计合成1对引物，扩增SjMP10 DNA片段并克隆到表达载体pGEX-4T-3中。转化BL21(DE3)后，诱导表达谷胱甘肽转移酶-虫卵中毛蚴抗原10（GST-SjMP10）融合蛋白。采用洗脱法制备GST-SjMP10融合蛋白，应用免疫印迹法（Western blotting)和淋巴细胞增殖试验进行重组蛋白的抗原性分析。 结果 SjMP10基因克隆到表达质粒pGEX-4T-3后，经异丙基-β-D-硫代半乳糖苷(IPTG)诱导能表达出Mr 39 000的GST-SjMP10融合蛋白，经电洗脱法纯化的上述重组融合蛋白可被血吸虫感染兔血清所识别，并能刺激血吸虫感染鼠脾淋巴细胞增殖。 结论 日本血吸虫虫卵中毛蚴抗原SjMP10融合表达获得成功。     

Immunodiagnosis of human eosinophilic meningitis using an antigen of Angiostrongylus cantonensis L5 with molecular weight 204 kD

An antigen from Angiostrongylus cantonensis fifth-stage larvae was purified by immuno-affinity chromatography with a specific monoclonal antibody. The purified antigen showed only a single band with a molecular weight of 204 kD in SDS-PAGE, and no cross-reactivity to antibodies induced by several other species of helminths were observed in ELISA. When the purified antigen was used to examine serum and cerebrospinal fluid (CSF) specimens by ELISA, the antibody levels in patients with eosinophilic meningitis or meningoencephalitis (EME) were significantly higher than those of control subjects. The antibody levels in serum were slightly higher than those in CSF, and the levels in serum were positively correlated with the levels in CSF. The reliability in detection of antibodies in serum was slightly higher than that in the detection of antibodies in CSF specimens. The purification of a specific A. cantonensis antigen and its subsequent use in the development of an ELISA for detection of A. cantonensis specific antibodies in serum specimens constitute an important step towards improvement in the accuracy of diagnosis for A. cantonensis infections.     

Protease secreted by the infective larvae of angiostrongylus cantonensis and its role in the penetration of mouse intestine

The infective third-stage larvae of Angiostrongylus cantonensis secrete a proteolytic enzyme that is thought to be essential for both larval penetration into the intestinal wall of the host and full development. Protease activity in these larvae during culture in vitro was determined by zymography, pH optimum, and substrate and inhibitor specificity. Excretory-secretory (ES) products of the third-stage larvae showed protease activity as three bands with molecular masses of 66, 30, and 23 kD by gelatin zymography. The optimal pH value for this protease activity was 10.0. The protease was found to have collagenolytic as well as elastinolytic activity, but these activities were inhibited by serine protease or metalloprotease inhibitors. The importance of this protease in larval penetration of the intestinal wall and entering the blood stream was observed in vitro by cocultured third-stage larvae of A. cantonensis with specific protease inhibitors in the intestines of BALB/c mice. The penetration rates of larvae significantly decreased when serine protease or metalloprotease inhibitors were added to the intestines. These results showed that serine protease and metalloprotease in ES products of A. cantonensis third-stage larvae are associated with larval penetration of the intestinal walls of mice.     

广州管圆线虫成虫cDNA文库抗原基因的筛选

目的从广州管圆线虫成虫cDNA文库中筛选特异性抗原基因。方法用大鼠感染血清做免疫探针,筛选广州管圆线虫成虫cDNA文库,对筛选得到的阳性克隆作交叉反应试验,PCR扩增外源基因插入片段并测序,用在线生物信息学软件进行同源性比对,推导氨基酸序列和蛋白质结构及进行功能分析。结果获得15个阳性克隆,分属三种基因,1个属中间纤维(IF)家族基因,3个与旋盘尾丝虫和秀丽杆线虫的真皮抗原同源,11个属主要精原蛋白(MSP)家族基因。其中1种基因表达产物没有发生明显交叉反应。结论从广州管圆线虫成虫cDNA文库中初步筛选出1个潜在特异性抗原基因和1个可能的主要抗原基因。     

金标法检测广州管圆线虫特异性IgG抗体的研究

目的探索简易、快速的金标免疫渗滤法检测广州管圆线虫特异性抗体的诊断价值。方法以广州管圆线虫成虫可溶性抗原点样于固相硝酸纤维素膜上，以胶体金-proteinA为标记物，采用自行设计的渗滤装置，检测病人血清中广州管圆线虫特异性IgG抗体。结果检测21人份广州管圆线虫病人血清，检出阳性19份，敏感性为90．5％（19／21）；100人份健康人群血清2份阳性，假阳性率为2％（2／100）；与旋毛虫病人、血吸虫病人血清交叉反应率分别为26．7％（8／30）、3．3％（1／30），未发现与肺吸虫、囊虫、华支睾吸虫、肺结核病人血清有交叉反应。结论金标免疫渗滤法快速检测血清中广州管圆线虫特异性IgG不仅操作简便、快速，结果判读容易，不需特殊仪器设备，而且敏感性高，有较好的辅助诊断价值，与旋毛虫病人血清存在交叉反应。     

广州管圆线虫组织蛋白酶Z基因的原核表达及免疫学分析

目的克隆并原核表达广州管圆线虫组织蛋白酶-Z基因,评价其融合蛋白在免疫诊断中的应用前景。方法利用生物信息学分析工具,分析广州管圆线虫组织蛋白酶-Z的理化性质、结构与功能特征;克隆目的基因至原核表达载体pET30a(+),经PCR、双酶切鉴定后,IPTG诱导表达,表达产物通过SDS-PAGE鉴定,融合蛋白用His-镍蛋白纯化柱纯化;ELISA检测融合蛋白作为诊断抗原的敏感性及特异性。结果该蛋白理化性质较稳定,含有分泌型信号肽;含有构成半胱氨酸蛋白酶催化中心的Cys84、His232和Asn253三个氨基酸残基。成功构建了重组质粒且目的基因在E.coliBL21中获得高效表达,经亲和层析获得了纯化的融合蛋白。融合蛋白可被其免疫的BALB/c小鼠血清及感染广州管圆线虫的小鼠血清识别;作为包被抗原用于ELISA检测小鼠血清其敏感性及特异性均为100%与粗抗原无差别,检测其他寄生虫病人血清及正常人血清其特异性分别为100%和97.4%与粗抗原相比特异性较高。结论广州管圆线虫组织蛋白酶-Z与多个物种组织蛋白酶-Z基因同源,是一种半胱氨酸蛋白酶,含有信号肽,可能是重要的虫体分泌排泄抗原成分,在广州管圆线虫病的免疫诊断方面有潜在的应用前景。     

Common antigenicities between Angiostrongylus cantonensis and various species of snails

Studies on common antigenicities were carried out by using rabbit sera immunized with Angiostrongylus cantonensis adult worms or the third stage larvae and antigens of various species of snails and vice versa by the immunoblotting technique. The results obtained are summarized as follows: (1) Common antigenicities between A. cantonensis adult worms and snails susceptible to A. cantonensis were observed in a range of molecular weights of 14.3 to 200 kDa. In Puerto Rican pigmented Biomphalaria glabrata and Achatina fulca, which had high infection rates with A. cantonensis, we recognized 15 to 16 bands against the adult worm, especially the band with a molecular weight of 29 kDa, which had a more intense reaction. (2) Common antigenicities between A. cantonensis third stage larvae and snails susceptible to A. cantonensis, were observed in a range of molecular weights of 14.3 to 97.4 kDa, especially A. fulica and B. glabrata, where we detected many bands in molecular weight range of 18.4 to 43 kDa. Based on the common antigenicities between A. cantonensis and snails susceptible to A. cantonensis, it is possible that the common antigenicities are one of the factors defining the different susceptibilities of various species of snails to A. cantonensis, and more bands are seen with increasing infection rates with A. cantonensis. Of those bands, the protein with the molecular weight of 29 kDa may be the main common antigen between the A. cantonensis adult worm, the third stage larvae and the snails susceptible to A. cantonensis.     

Local antibody production and immune complex formation in rats experimentally infected with Angiostrongylus cantonensis

The systemic humoral immune responses and tissue localization of worm-antigen, antibodies (IgG), and complement (C3) were examined in rats experimentally infected with Angiostrongylus cantonensis. While the worms remained in the subarachnoid space, it was infiltrated with plasma cells and lymphoid cells containing IgM and IgG. When the infiltration of these cells became more pronounced, the serum antibody titer began to increase. At the same time, deposits of IgM, IgG, and C3 were found in the glomeruli of the kidney. A number of eggs were observed in the lungs, enclosed in granulomatous tissues. Infiltrates of plasma cells including IgM and IgG, and deposits of IgM, IgG, and C3 were detected around the eggs and in the granulomatous tissues. A marked increase in serum antibody was observed. A. cantonensis larvae induce local antibody (IgM and IgG) production in the central nervous system prior to an increase of serum antibody titer. Measurement of cerebrospinal fluid antibody titer at an early stage of infection may confirm infection. The larvae showed no evidence of damage in spite of marked local antibody production in the central nervous system. The eggs in the lungs stimulated both local and systemic antibody production, and immune complexes were formed in the lung and the circulatory system. Immune complexes may participate in the formation of granuloma.