【摘要】： 目的鉤蟲病是全球發(fā)展中國家常見的腸道寄生蟲病,反復藥物驅蟲導致耐藥危險性增加,而藥物的毒副作用也限制了化療藥物在孕婦及嬰兒患者中的使用。因此,單純依靠驅蟲藥物的防治方法已經(jīng)不能完全適應現(xiàn)階段全球鉤蟲病的防治工作,安全、有效的抗鉤蟲病替代藥物或者功能性食品有待于研制。本課題收集鉤蟲病患者驅蟲后排出的鉤蟲成蟲,制備鉤蟲成蟲抗原(AWA),免疫25w產(chǎn)蛋海蘭母雞,制備抗鉤蟲成蟲特異性卵黃抗體(IgY)并鑒定。動態(tài)監(jiān)測抗體效價,大量純化效價較高階段IgY,通過體內(nèi)、體外試驗分析特異性IgY的抗蚴作用,為進一步研制抗鉤蟲藥物或功能性食品奠定基礎。 方法應用飽和鹽水漂浮法加水洗沉淀法篩查安徽醫(yī)科大學第一附屬醫(yī)院住院患者23人,收集4位糞檢陽性患者藥物驅蟲后1-5d全程糞便,水洗過篩收集成蟲,制備AWA保存?zhèn)溆谩Ｙ徺I未經(jīng)免疫的海蘭母雞雛雞,養(yǎng)至25W產(chǎn)蛋。每只母雞以200ugAWA進行皮下加肌肉多點注射免疫,初次免疫后28d給予第二次免疫,間隔10d進行第三次免疫。收集免疫前、后雞蛋,標記后4℃保存?zhèn)溆�。水稀釋法粗提抗鉤蟲IgY抗體,鹽析法純化抗體,SDS-PAGE和Western-blotting進行鑒定。間接ELISA法測定抗體效價,選取效價最高時間段的雞蛋,大量提取純化IgY抗體4℃保存?zhèn)溆谩２捎酶牧技犹俸裢科?Kato-Katz法)檢獲安徽省蒙城縣重度鉤蟲感染患者,收集患者糞便,分別進行固體培養(yǎng)基濾紙法鉤蚴培養(yǎng)和試管濾紙法鉤蚴培養(yǎng)。分別收集鉤蟲三期幼蟲(L3),進行體外L3期鉤蚴與30%,50%和70%濃度的IgY抗體分別共培養(yǎng)以及小鼠腹腔內(nèi)L3期鉤蚴與IgY抗體共培養(yǎng)。倒置顯微鏡下觀察體外共培養(yǎng)后鉤蚴形態(tài),并進行鉤蚴存活率統(tǒng)計分析。透射電鏡觀察小鼠腹腔內(nèi)抗體共培養(yǎng)鉤蚴形態(tài),倒置顯微鏡下根據(jù)中性粒細胞黏附程度的不同對鉤蚴進行分級計數(shù),統(tǒng)計學分析。利用IgY抗體和FITC標記的抗雞IgY二抗進行間接免疫熒光實驗,觀察鉤蟲成蟲抗原在鉤蚴體表的分布特點。 結果制備的鉤蟲AWA濃度為1.21mg/ml。水稀釋粗提、鹽析法純化得到的IgY,經(jīng)Lowry法測得的濃度為1.8mg/ml。SDS-PAGE檢測IgY分子量與理論值一致。Western-blotting顯示,免疫后的IgY與AWA可以相互識別,而免疫前的IgY則不能識別成蟲抗原蛋白。間接ELISA法測定抗體效價,初次免疫后10d左右產(chǎn)生抗體,隨著時間推移,抗體滴度逐漸上升,至免疫后55d達到最高峰,效價達到1:10000以上。不同產(chǎn)蛋母雞之間抗體滴度存在個體差異。用EGG stractTM IgY Purification System純化試劑盒大量純化IgY,經(jīng)Lowry法測得的濃度在5.9-10.1mg/ml之間。鉤蚴與IgY體外共培養(yǎng),鉤蚴體表形成絮狀或顆粒狀附著物,絮狀物纏繞蟲體,使蟲體活動受限;鉤蚴存活率統(tǒng)計學分析表明:50%及70%濃度的IgY抗體能夠對鉤蚴的存活產(chǎn)生影響。小鼠腹腔內(nèi)鉤蚴與抗體共培養(yǎng)顯示,IgY抗體能夠增加中性粒細胞對鉤蚴的趨化、黏附作用。透射電鏡觀察發(fā)現(xiàn),蟲體表膜腫脹、粗糙,橫紋間相互的擠壓,表膜及肌層電子密度不均勻。免疫熒光實驗顯示,鉤蚴表膜上存在著與鉤蟲成蟲相同的抗原組分,利用成蟲粗抗原免疫母雞得到的IgY抗體可與鉤蚴表膜上的抗原結合。 結論本研究成功大量制備了抗鉤蟲成蟲特異性IgY抗體,具有良好的安全性和穩(wěn)定性,易于保存。我們發(fā)現(xiàn)IgY抗體可與鉤蚴表膜抗原結合,能夠增加中性粒細胞對鉤蚴的趨化、黏附作用;IgY抗體對蟲體表膜有損傷作用,高濃度IgY抗體影響鉤蚴的存活率。表明,特異性IgY抗體對鉤蚴具有損傷作用,可用于鉤蟲病的被動免疫。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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