抗钩虫特异性卵黄抗体的制备与鉴定及其抗钩蚴作用研究

发布时间：2018-09-10 08:00

【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。
[Abstract]:Objective Ancylosis is a common intestinal parasitic disease in developing countries. Repeated drug repellent treatment increases the risk of drug resistance, and the side effects of drugs restrict the use of chemotherapy drugs in pregnant women and infants. A safe and effective alternative drug or functional food for the treatment of hookworm disease needs to be developed.The adults of hookworm discharged from the patients with hookworm disease after treatment were collected to prepare hookworm adult worm antigen (AWA) and immunize 25-week laying hens to prepare specific egg yolk antibodies (IgY) against hookworm adults and identify them.The titers of antibodies were monitored dynamically and purified in large quantities. In vitro and in vivo tests were carried out to analyze the anti-metacercariae effect of specific IgY with high titer IgY, so as to lay a foundation for further development of anti-hookworm drugs or functional foods.
Methods Twenty-three inpatients in the First Affiliated Hospital of Anhui Medical University were screened by the method of saturated saline floatation and water-washing precipitation. The stools of four positive patients were collected 1-5 days after drug-repellent treatment. The adults were washed and collected to prepare AWA for preservation. The eggs were collected before and after immunization and stored at 4 C after labeling. Anti-hookworm IgY antibody was crude extracted by water dilution method, purified by salting-out method, identified by SDS-PAGE and Western-blotting. The titer of antibody was determined by indirect ELISA and selected. The eggs with the highest titer of IgY were extracted and purified in large quantities and stored at 4 C. The feces of patients with severe hookworm infection in Mengcheng County of Anhui Province were collected by modified Kato-Katz method. (L3) co-cultured with 30%, 50% and 70% IgY antibodies in vitro and co-cultured with IgY antibodies in mouse abdominal cavity respectively. The morphology of hookworm larvae was observed under inverted microscope and the survival rate of hookworm larvae was statistically analyzed. In order to observe the distribution of the antigens on the surface of leptospira, the IgY antibody and FITC labeled anti-chicken IgY antibody were used for indirect immunofluorescence assay.
Results The AWA concentration of hookworm was 1.21 mg/ml. The IgY purified by water dilution and salting-out was 1.8 mg/ml. The molecular weight of IgY detected by SDS-PAGE was consistent with the theoretical value. Western-blotting showed that the IgY and AWA could recognize each other after immunization, but the IgY before immunization could not recognize the adult antigen protein. The titer of antibody increased gradually with the passage of time, and reached the peak at 55 days after immunization. The titer of antibody varied with individual laying hens. IgY was purified by EGG stractTM IgY Purification System and measured by Lowry method. The concentration of IgY was between 5.9 mg/ml and 10.1 mg/ml. In vitro co-culture of Leptospira with IgY, a flocculent or granular attachment was formed on the surface of leptospira. The flocculent entanglement restricted the activity of leptospira. Statistical analysis of the survival rate of Leptospira showed that 50% and 70% IgY antibodies could affect the survival of leptospira. Y antibody can increase the chemotaxis and adhesion of neutrophils to hookworm larvae. Transmission electron microscopy showed that the surface membrane of hookworm larvae was swollen and rough, and the electron densities of the surface membrane and muscular layer were uneven. Immunofluorescence test showed that there were the same antigen components on the surface membrane of hookworm larvae as that of hookworm adults. The obtained IgY antibody can bind to the antigen on the membrane of the larvae.
Conclusion A large number of specific IgY antibodies against hookworm adults have been successfully prepared, which are safe, stable and easy to preserve. We found that IgY antibodies can bind to the surface membrane antigens of hookworm larvae and increase the chemotaxis and adhesion of neutrophils to hookworm larvae. The results showed that the specific IgY antibody could damage hookworm larvae and could be used for passive immunization of hookworm disease.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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