【作者】 王圆圆；

【导师】 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2010， 硕士

【摘要】 目的钩虫病是全球发展中国家常见的肠道寄生虫病,反复药物驱虫导致耐药危险性增加,而药物的毒副作用也限制了化疗药物在孕妇及婴儿患者中的使用。因此,单纯依靠驱虫药物的防治方法已经不能完全适应现阶段全球钩虫病的防治工作,安全、有效的抗钩虫病替代药物或者功能性食品有待于研制。本课题收集钩虫病患者驱虫后排出的钩虫成虫,制备钩虫成虫抗原(AWA),免疫25w产蛋海兰母鸡,制备抗钩虫成虫特异性卵黄抗体(IgY)并鉴定。动态监测抗体效价,大量纯化效价较高阶段IgY,通过体内、体外试验分析特异性IgY的抗蚴作用,为进一步研制抗钩虫药物或功能性食品奠定基础。方法应用饱和盐水漂浮法加水洗沉淀法筛查安徽医科大学第一附属医院住院患者23人,收集4位粪检阳性患者药物驱虫后1-5d全程粪便,水洗过筛收集成虫,制备AWA保存备用。购买未经免疫的海兰母鸡雏鸡,养至25W产蛋。每只母鸡以200ugAWA进行皮下加肌肉多点注射免疫,初次免疫后28d给予第二次免疫,间隔10d进行第三次免疫。收集免疫前、后鸡蛋,标记后4℃保存备用。水稀释法粗提抗钩虫IgY抗体,盐析法纯化抗体,SDS-PAGE和Western-blotting进行鉴定。间接ELISA法测定抗体效价,选取效价最高时间段的鸡蛋,大量提取纯化IgY抗体4℃保存备用。采用改良加藤厚涂片法(Kato-Katz法)检获安徽省蒙城县重度钩虫感染患者,收集患者粪便,分别进行固体培养基滤纸法钩蚴培养和试管滤纸法钩蚴培养。分别收集钩虫三期幼虫(L3),进行体外L3期钩蚴与30%,50%和70%浓度的IgY抗体分别共培养以及小鼠腹腔内L3期钩蚴与IgY抗体共培养。倒置显微镜下观察体外共培养后钩蚴形态,并进行钩蚴存活率统计分析。透射电镜观察小鼠腹腔内抗体共培养钩蚴形态,倒置显微镜下根据中性粒细胞黏附程度的不同对钩蚴进行分级计数,统计学分析。利用IgY抗体和FITC标记的抗鸡IgY二抗进行间接免疫荧光实验,观察钩虫成虫抗原在钩蚴体表的分布特点。结果制备的钩虫AWA浓度为1.21mg/ml。水稀释粗提、盐析法纯化得到的IgY,经Lowry法测得的浓度为1.8mg/ml。SDS-PAGE检测IgY分子量与理论值一致。Western-blotting显示,免疫后的IgY与AWA可以相互识别,而免疫前的IgY则不能识别成虫抗原蛋白。间接ELISA法测定抗体效价,初次免疫后10d左右产生抗体,随着时间推移,抗体滴度逐渐上升,至免疫后55d达到最高峰,效价达到1:10000以上。不同产蛋母鸡之间抗体滴度存在个体差异。用EGG stractTM IgY Purification System纯化试剂盒大量纯化IgY,经Lowry法测得的浓度在5.9-10.1mg/ml之间。钩蚴与IgY体外共培养,钩蚴体表形成絮状或颗粒状附着物,絮状物缠绕虫体,使虫体活动受限;钩蚴存活率统计学分析表明:50%及70%浓度的IgY抗体能够对钩蚴的存活产生影响。小鼠腹腔内钩蚴与抗体共培养显示,IgY抗体能够增加中性粒细胞对钩蚴的趋化、黏附作用。透射电镜观察发现,虫体表膜肿胀、粗糙,横纹间相互的挤压,表膜及肌层电子密度不均匀。免疫荧光实验显示,钩蚴表膜上存在着与钩虫成虫相同的抗原组分,利用成虫粗抗原免疫母鸡得到的IgY抗体可与钩蚴表膜上的抗原结合。结论本研究成功大量制备了抗钩虫成虫特异性IgY抗体,具有良好的安全性和稳定性,易于保存。我们发现IgY抗体可与钩蚴表膜抗原结合,能够增加中性粒细胞对钩蚴的趋化、黏附作用;IgY抗体对虫体表膜有损伤作用,高浓度IgY抗体影响钩蚴的存活率。表明,特异性IgY抗体对钩蚴具有损伤作用,可用于钩虫病的被动免疫。更多还原

【Abstract】 Objective: Ancylostomiasis is the common intestinal parasitic diseases in developing countries all over the world. Since repeated deworming leads to a potential risk of drug resistance as well as the drug toxicity and side-effect limits the application of deworming drugs in pregnant woman and infant, the treament depending on anthelmintic can not satisfy the demands on preventing and curing global ancylostomiasis..The safe and effective substitute drug or functional food needs to be developed. In this study, we prepared the adult worm antigen(AWA) using the ancylostomes obtained from the patients’feces who had taken anthelmintic to expel the parasite. Then the ancylostome specific yolk immunoglobulin (IgY) was prepared and identified from the 25-week old HY-line hens immunized by AWA. We monitored the dynamic a ntibody titer of IgY,and purified the antibody at higher titer. The IgY was culture with the third-stage infective larvae(L3) in vitro and in vivo to investigate the effect of IgY upon ancylostome and to lay the foundation for the production of anti-hookworm drugs or functional foods.Methods: Twenty-three patients from the first affiliated hospital of AnHui medical university were screened by brine flotation and sedimentation method. The dejecta from 4 patients with positive kopr-test was collected for consecutive five days after treated with anthelmintic, and the ancylostomes were sieved to produce AWA. The 25 week-old egg-laying HY-line hens were immunized with 200μg AWA by subcutaneous injection and intramuscular injection.On the day 28th and the day 38th after initial immunization, the hens were immunized repeatedly.The eggs layed before and after immunization were collected to make anti-AWA IgY antibody(Storage Temperature is 4℃).IgY was extracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDS-PAGE and Western-blotting. Indirect ELISA was used to determine the titer of the antiserum. Large amount IgY was purified while the antibody had a high titer. The patients with severe ancylostomiasis infection in Mengcheng County, Anhui Province diagnosed by Kato-Katz were included in the following experiment. Their dejecta were cultured by solid medium-filter paper cultivation of ancylostoma caninum larvae and tube-filter paper cultivation and the L3 were isolated. The L3 was cultured with IgY at the concentration of 30%, 50% and 70% respectively in vitro or in the abdominal cavity of mice.The larvae cultured in vitro were observed by inverted microscope and their survival rate was calculated. The larvae obtained from the mice were observed by transmission electron microscopy(TEM) and graded according to the neutrophil adhesion by inverted microscope. The distribution of ancylostome antigen on L3 was observated by indirect immunofluorescence staining using anti-IgY and FITC conjugated chicken IgY antibody.Result: The concentration of AWA was 1.21mg/ml.The concentration of IgY obtained by Lowry method was 1.8mg/ml. The molecular size of IgY detected by SDS-PAGE was equal to its predictive value. Western blotting results showed that the AWA could be recognized by IgY collected from immunizsed hens while not by IgY collected before immunization. Using indirect ELISA, the facts were observed that the IgY antibody began to produce by hens on the tenth day after the initial immunization, and the antibody titer increased gradually over time while reached the peak with a titer overtoped 1:10000 on the 55th day after immunization. There is a discrepancy on IgY antibody titer between different hens. The IgY was purified heavy by EGG stractTMIgY Purification System and the concentration ranged from 5.9 mg/ml to 10.1mg/ml. A floc or granular attachment formed on the surface of L3 after culture in vitro. The activity of L3 had been restricted by attachments. Statistical analysis showed that the IgY with the concentration of 50% or 70% could decrease the livability of L3. It was found that the IgY antibodies could increase chemotaxis and adhesion of neutrophil through the in vivo experiment.TEM observation showed that L3 exhibited a damage of its surface including swelling, roughness, uneven electron density, and the transverse striations of L3 squeezed each other.A same antigen component of adult worm existed on the superficial membrane of L3 disclosed by indirect immunofluorescence.The purified IgY could react with antigen on the cuticular surface of L3.Conclusion: We have successfully prepared the IgY antibody specific to ancylostome with good security and stability. The yield of IgY is generous and easy to store.The IgY can react with antigen on cuticular surface of L3 and increase chemotaxis and adhesion effect of neutrophil. The IgY bined to L3 may damage its superficial membrane and the antibody with high concentration can decrease the livability of L3.In short, the specific IgY is harmful to larvae, and can be used for immunotherapy against ancylostomiasis.更多还原