筛选日本血吸虫童虫早期诊断抗原的研究

发布时间：2018-01-10 14:06

本文关键词：筛选日本血吸虫童虫早期诊断抗原的研究　出处：《武汉大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的采用十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)和酶联免疫印迹术(EITB)筛选日本血吸虫童虫早期诊断抗原分子。同时,利用噬菌体随机12肽库技术筛选日本血吸虫感染早期诊断抗原模拟表位,经动物实验评估其早期诊断价值,并观察其抗血吸虫感染的免疫保护效果。方法收集日本血吸虫童虫与成虫,制各可溶性抗原,进行SDS-PAGE电泳后,转印至硝酸纤维素膜上,分别与感染后10d、21d、42d及正常兔血清进行免疫应印渍试验,比较两者反应条带的差异性,筛选出能被早期感染血清所识别的抗原组分。用日本血吸虫感染后21d血清从噬菌体12肽库中经3轮筛选,有效地富集与早期感染兔血清有亲和力的噬菌体克隆。随机挑取11个单克隆分别纯化、扩增,随后采用ELISA检测方法,挑选出与早期感染兔血清有高亲和力的噬菌体克隆,经动物实验鉴定其对日本血吸虫感染的早期诊断效果。同时按1×10~(15)pfu/次剂量,0-2-4W方案,皮下多点注射免疫小鼠,末次免疫4W后,每鼠经腹部皮肤攻击感染40±1条日本血吸虫尾蚴,感染42d后观察减虫率及减卵率。结果日本血吸虫童虫可溶性抗原(SSA)和成虫可溶性抗原(SAWA)经SDS-PAGE后显示:SSA出现的条带数多于SAWA,主要位于分子量大于97kDa的区域,其它区域条带数及各条带迁移率大致相同,为期共同抗原。将SSA和SAWA分别与感染前及感染后10d、21d、42d兔血清进行EITB试验结果显示:SSA和SAWA与感染前兔血清均未出现反应带;与SAWA相比较,SSA的早期反应抗原带较多较强,SSA共出现12条反应带,其中97kDa和47kDa抗原分子只能被感染后10d兔血清特异性识别,而不能被感染后21d、42d兔血清识别,140kDa、107kDa、87kDa、74kDa、67kDa、62kDa、44kDa、32kDa、26kDa、16kDa抗原分子均能被感染后10d、21d、42d兔血清识别,除32kDa和16kDa与感染21d兔血清反应较弱外,其他抗原分子的反应强度随感染天数逐渐增强;SAWA仅出现7条反应带,未显示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 卤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
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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