本文關(guān)鍵詞：篩選日本血吸蟲童蟲早期診斷抗原的研究　出處：《武漢大學》2005年碩士論文　論文類型：學位論文

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【摘要】：目的 采用十二烷基磺酸鈉—聚丙烯酰胺凝膠電泳(SDS-PAGE)和酶聯(lián)免疫印跡術(shù)(EITB)篩選日本血吸蟲童蟲早期診斷抗原分子。同時,利用噬菌體隨機12肽庫技術(shù)篩選日本血吸蟲感染早期診斷抗原模擬表位,經(jīng)動物實驗評估其早期診斷價值,并觀察其抗血吸蟲感染的免疫保護效果。 方法 收集日本血吸蟲童蟲與成蟲,制各可溶性抗原,進行SDS-PAGE電泳后,轉(zhuǎn)印至硝酸纖維素膜上,分別與感染后10d、21d、42d及正常兔血清進行免疫應印漬試驗,比較兩者反應條帶的差異性,篩選出能被早期感染血清所識別的抗原組分。用日本血吸蟲感染后21d血清從噬菌體12肽庫中經(jīng)3輪篩選,有效地富集與早期感染兔血清有親和力的噬菌體克隆。隨機挑取11個單克隆分別純化、擴增,隨后采用ELISA檢測方法,挑選出與早期感染兔血清有高親和力的噬菌體克隆,經(jīng)動物實驗鑒定其對日本血吸蟲感染的早期診斷效果。同時按1×10~(15)pfu/次劑量,0-2-4W方案,皮下多點注射免疫小鼠,末次免疫4W后,每鼠經(jīng)腹部皮膚攻擊感染40±1條日本血吸蟲尾蚴,感染42d后觀察減蟲率及減卵率。 結(jié)果 日本血吸蟲童蟲可溶性抗原(SSA)和成蟲可溶性抗原(SAWA)經(jīng)SDS-PAGE后顯示:SSA出現(xiàn)的條帶數(shù)多于SAWA,主要位于分子量大于97kDa的區(qū)域,其它區(qū)域條帶數(shù)及各條帶遷移率大致相同,為期共同抗原。將SSA和SAWA分別與感染前及感染后10d、21d、42d兔血清進行EITB試驗結(jié)果顯示:SSA和SAWA與感染前兔血清均未出現(xiàn)反應帶;與SAWA相比較,SSA的早期反應抗原帶較多較強,SSA共出現(xiàn)12條反應帶,其中97kDa和47kDa抗原分子只能被感染后10d兔血清特異性識別,而不能被感染后21d、42d兔血清識別,140kDa、107kDa、87kDa、74kDa、67kDa、62kDa、44kDa、32kDa、26kDa、16kDa抗原分子均能被感染后10d、21d、42d兔血清識別,除32kDa和16kDa與感染21d兔血清反應較弱外,其他抗原分子的反應強度隨感染天數(shù)逐漸增強;SAWA僅出現(xiàn)7條反應帶,未顯示140kDa、107kDa、97kDa、62kDa和
[Abstract]:Objective to screen the antigens for early diagnosis of schistosomiasis japonicum by SDS-PAGE and EITB using sodium 12 alkyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunoblotting (Elisa). The mimic epitopes of early diagnosis antigen of schistosomiasis japonicum infection were screened by phage random 12 peptide library technique. The value of early diagnosis was evaluated by animal experiments, and the immune protection effect of phage random 12 peptide library against schistosomiasis infection was observed. Methods Schistosoma japonicum and adult Schistosoma japonicum were collected and the soluble antigens were prepared. After SDS-PAGE electrophoresis, they were transferred to the nitrocellulose membrane for 10 days and 21 days after infection. The difference of the reaction bands was compared between the 42 days and the normal rabbit serum. The antigen components which could be recognized by early infection serum were screened from phage 12 peptide library by three rounds with the serum of Schistosoma japonicum 21 days after infection. The phage clones with affinity to early infected rabbit serum were effectively enriched. 11 monoclonal clones were randomly selected for purification, amplification and ELISA detection. The phage clones with high affinity to the sera of early infected rabbits were selected, and the early diagnosis of Schistosoma japonicum infection was confirmed by animal experiments. Mice were immunized with 0-2-4W regimen. After the last immunization for 4W, each mouse was infected with 40 鹵1 cercariae of Schistosoma japonicum through abdominal skin. The worm reduction rate and egg reduction rate were observed after 42 days of infection. Results SDS-PAGE showed that there were more bands of SDS-PAGE in Schistosoma japonicum than in SAWA. It was mainly located in the region with molecular weight greater than 97kDa. The number of bands and the mobility of each band in other regions were about the same. The SSA and SAWA were compared with those before infection and 10 days after infection, respectively. The results of EITB test showed that there were no reaction bands between the two groups before and after infection. Compared with SAWA, the early reaction antigen bands of SSA were much stronger than that of SSA, and there were 12 reaction bands in SSA. Among them, 97 kDa and 47 kDa antigen molecules could only be specifically identified by rabbit serum on 10 days after infection, but not by 140 kDa or 107 kDa in rabbit serum on 21 and 42 days after infection. 87kDa74kDaO67kDa62kDa4kDa44kDa32kDa26kDa16kDa antigen molecules can be infected for 10 days and 21 days after infection. In 42-day rabbit serum recognition, the reaction intensity of other antigenic molecules increased gradually with the days of infection, except that 32kDa and 16kDa reacted weakly to the sera of 21-day rabbits. There were only 7 reaction bands in SAWA, but no 140kDaA 107kDa97kDa 62kDa and
【學位授予單位】：武漢大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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