【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(fù)藥物驅(qū)蟲導(dǎo)致耐藥危險(xiǎn)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(qū)蟲藥物的防治方法已經(jīng)不能完全適應(yīng)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(qū)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(dòng)態(tài)監(jiān)測(cè)抗體效價(jià),大量純化效價(jià)較高階段IgY,通過體內(nèi)、體外試驗(yàn)分析特異性IgY的抗蚴作用,為進(jìn)一步研制抗鉤蟲藥物或功能性食品奠定基礎(chǔ)。 方法應(yīng)用飽和鹽水漂浮法加水洗沉淀法篩查安徽醫(yī)科大學(xué)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(qū)蟲后1-5d全程糞便,水洗過篩收集成蟲,制備AWA保存?zhèn)溆�。購買未經(jīng)免疫的海蘭母雞雛雞,養(yǎng)至25W產(chǎn)蛋。每只母雞以200ugAWA進(jìn)行皮下加肌肉多點(diǎn)注射免疫,初次免疫后28d給予第二次免疫,間隔10d進(jìn)行第三次免疫。收集免疫前、后雞蛋,標(biāo)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(jìn)行鑒定。間接ELISA法測(cè)定抗體效價(jià),選取效價(jià)最高時(shí)間段的雞蛋,大量提取純化IgY抗體4℃保存?zhèn)溆�。采用改良加藤厚涂片�?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(jìn)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(jìn)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(jìn)行鉤蚴存活率統(tǒng)計(jì)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(xì)胞黏附程度的不同對(duì)鉤蚴進(jìn)行分級(jí)計(jì)數(shù),統(tǒng)計(jì)學(xué)分析。利用IgY抗體和FITC標(biāo)記的抗雞IgY二抗進(jìn)行間接免疫熒光實(shí)驗(yàn),觀察鉤蟲成蟲抗原在鉤蚴體表的分布特點(diǎn)。 結(jié)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(cè)得的濃度為1.8mg/ml。SDS-PAGE檢測(cè)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(shí)別,而免疫前的IgY則不能識(shí)別成蟲抗原蛋白。間接ELISA法測(cè)定抗體效價(jià),初次免疫后10d左右產(chǎn)生抗體,隨著時(shí)間推移,抗體滴度逐漸上升,至免疫后55d達(dá)到最高峰,效價(jià)達(dá)到1:10000以上。不同產(chǎn)蛋母雞之間抗體滴度存在個(gè)體差異。用EGG stractTM IgY Purification System純化試劑盒大量純化IgY,經(jīng)Lowry法測(cè)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(dòng)受限;鉤蚴存活率統(tǒng)計(jì)學(xué)分析表明:50%及70%濃度的IgY抗體能夠?qū)︺^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(xì)胞對(duì)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(shí)驗(yàn)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(jié)合。 結(jié)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(jié)合,能夠增加中性粒細(xì)胞對(duì)鉤蚴的趨化、黏附作用;IgY抗體對(duì)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(duì)鉤蚴具有損傷作用,可用于鉤蟲病的被動(dòng)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(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 藺西萌，李桂蓮，姚黨生;河南省鉤蟲病流行病學(xué)調(diào)查[J];疾病監(jiān)測(cè);1996年01期

2 謝永強(qiáng),張進(jìn)鋼,侯鳳玲;鉤蟲病引起上消化道大出血一例報(bào)告[J];北京醫(yī)學(xué);2003年04期

3 王芳,周萍,韋統(tǒng)友;宮內(nèi)感染性鉤蟲病一例誤診[J];臨床誤診誤治;1999年01期

4 羅同仁,桂正平,李繼銀,楊生碧,陳應(yīng)瓊;阿苯達(dá)唑防治鉤蟲病的效果[J];中國寄生蟲學(xué)與寄生蟲病雜志;1994年S1期

5 任碧清,王天順,陶斯象;四川廣安農(nóng)村人群鉤蟲病感染和患病情況調(diào)查[J];川北醫(yī)學(xué)院學(xué)報(bào);1997年04期

6 范士芹;56例鉤蟲病臨床漏診誤診分析[J];農(nóng)墾醫(yī)學(xué);2000年03期

7 張栓龍,王啟勝,李華斌;鉤蟲病致消化道大出血誤診剖腹探查一例[J];中華內(nèi)科雜志;1996年10期

8 楊兆莘;陳啟仁;;有關(guān)鉤蚴培養(yǎng)幾種因素的探討[J];安徽醫(yī)科大學(xué)學(xué)報(bào);1961年Z1期

9 羅平;鉤蟲病重度貧血誤診為精神分裂癥1例[J];四川醫(yī)學(xué);2005年05期

10 莊火金,方明亮,陳蕉香,許傳教,朱瓊光,郭婉秋,林峰;云霄縣鉤蟲病流行病學(xué)調(diào)查[J];中國血吸蟲病防治雜志;1992年04期

相關(guān)會(huì)議論文 前10條

1 楊維平;;鉤蟲病防治研究進(jìn)展[A];全國新出現(xiàn)傳染病學(xué)術(shù)研討會(huì)資料匯編[C];2004年

2 黃敏君;孫嵐;郭增柱;;鉤蟲病致上消化道出血一例報(bào)道[A];中華醫(yī)學(xué)會(huì)第五次全國艾滋病、病毒性丙型肝炎暨全國熱帶病學(xué)術(shù)會(huì)議論文匯編[C];2011年

3 顏小瓊;;鉤蟲病88例報(bào)道[A];中華醫(yī)學(xué)會(huì)第十三屆全國放射學(xué)大會(huì)論文匯編（下冊(cè)）[C];2006年

4 陳佳敏;蔡建庭;陳焰;鐘丹丹;杜勤;陳妙研;;OMOM膠囊內(nèi)鏡診斷小腸鉤蟲病2例[A];首屆浙江省消化病學(xué)術(shù)大會(huì)論文匯編[C];2008年

5 楊維平;;鉤蟲病防治研究進(jìn)展[A];全國EID研討班資料匯編[C];2004年

6 余培珍;林兆京;林木水;;群養(yǎng)狼犬暴發(fā)鉤蟲病[A];福建省家畜寄生蟲病學(xué)會(huì)第三次學(xué)術(shù)討論會(huì)論文選（1996）[C];1996年

7 毛天翔;張忠傳;;犬出血性腸炎的綜合診斷與治療[A];中國畜牧獸醫(yī)學(xué)會(huì)養(yǎng)犬學(xué)分會(huì)第十二次全國養(yǎng)犬學(xué)術(shù)研討會(huì)論文集[C];2007年

8 蔣詩國;肖邦忠;羅興建;羅飛;吳成果;李繼艮;肖鵬;黃文利;;重慶市人群土源性寄生蟲感染趨勢(shì)與感染因素分析[A];重慶市預(yù)防醫(yī)學(xué)會(huì)2009年論文集[C];2009年

9 馮文才;;疑難病案二則[A];第四屆全國疑難病學(xué)術(shù)研討會(huì)資料匯編[C];2006年

10 張弟;;成都市1996～1998年鉤蟲病防治工作的經(jīng)濟(jì)效益分析[A];全面建設(shè)小康社會(huì)：中國科技工作者的歷史責(zé)任——中國科協(xié)2003年學(xué)術(shù)年會(huì)論文集（上）[C];2003年

相關(guān)重要報(bào)紙文章 前10條

1 韓振奎;起糞肥注意防“鉤蚴”[N];陜西科技報(bào);2009年

2 ;當(dāng)心鉤蟲病染上身[N];中國醫(yī)藥報(bào);2005年

3 文/梁良;防治肉犬鉤蟲病[N];山西科技報(bào);2001年

4 山東省寄生蟲病防治研究所 于振華;當(dāng)心鉤蟲病“鉤”去你的幸福生活[N];大眾衛(wèi)生報(bào);2006年

5 榮雯;蟯蟲、鉤蟲病家庭簡(jiǎn)易療法[N];大眾衛(wèi)生報(bào);2003年

6 錢培康;當(dāng)心糞肥惹“糞毒”[N];健康報(bào);2006年

7 韓振奎;春耕在前 防病當(dāng)先[N];大眾衛(wèi)生報(bào);2008年

8 羅空;污染土壤對(duì)人體健康的影響[N];農(nóng)村醫(yī)藥報(bào)（漢）;2009年

9 李莉;農(nóng)民要防職業(yè)病[N];中國特產(chǎn)報(bào);2003年

10 ;解開蟲蟲致病之謎[N];醫(yī)藥經(jīng)濟(jì)報(bào);2005年

相關(guān)碩士學(xué)位論文 前6條

1 王圓圓;抗鉤蟲特異性卵黃抗體的制備與鑒定及其抗鉤蚴作用研究[D];安徽醫(yī)科大學(xué);2010年

2 趙瑋;明清以來珠江三角洲地區(qū)的“�；~塘”與鉤蟲病[D];中國海洋大學(xué);2008年

3 楊杜鵑;1642例膠囊內(nèi)鏡檢查患者的回顧性分析[D];浙江大學(xué);2011年

4 房功思;溶血磷脂酶Sj1539蛋白在日本血吸蟲病免疫學(xué)診斷中的初步應(yīng)用[D];安徽醫(yī)科大學(xué);2011年

5 陳偉嬋;297例OMOM膠囊內(nèi)鏡臨床應(yīng)用研究[D];重慶醫(yī)科大學(xué);2012年

6 蔡文聘;小腸出血的病因及臨床診斷分析[D];福建醫(yī)科大學(xué);2012年

，

本文編號(hào)：2233850