人源日本血吸虫Fab抗体库的构建及抗独特型抗体的筛选、鉴定
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国家自然科学基金资助(30371259)


Screening and identification of a human Fab antibody against idiotypic antibody from a phage-display library of schistosoma japonicum
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    摘要:

    目的:构建人源日本血吸虫Fab抗体库,筛选出日本血吸虫抗独特型抗体并进行初步鉴定。方法: 以3名晚期血吸虫患者切除的脾脏为源,构建人源日本血吸虫Fab抗体库。并以日本血吸虫可溶性虫卵抗原(SEA)及成虫抗原(SWAP)免疫鼠血清IgG包板进行筛选,经ELISA鉴定后,对阳性克隆进行可溶性表达。结果:构建了人源日本血吸虫Fab抗体库,库容为4.3 × 106,经核苷酸序列分析,证实插入片段为Fab基因片断。经过5轮筛选后,从富集的次级抗体库中随机挑取60个克隆,用ELISA法鉴定得到4个可与免疫鼠血清(Ab1)特异性结合,而不与SEA及SWAP结合的单克隆抗体(A3、B6、C4、C17),其中B6经SDS-PAGE电泳显示插入片断正确。结论:成功构建了人源日本血吸虫Fab抗体库, 并从中获得人源日本血吸虫抗独特型抗体B6,为研制用于人体血吸虫病免疫预防的抗独特型抗体疫苗奠定了基础。

    Abstract:

    Objective: To obtain the completely humanized Fab antibody against idiotypic antibody from a phage-display library of schistosoma japonicum. Methods: A schistosoma japonicum library of phage-display human Fab library was constructed using genes from spleens of 3 patients with schistosoma japonicum infection. Soluble egg antigen(SEA) and soluble worm antigen preparations (SWAP) were used to screen schistosoma japonicum immune mouse serum IgG. The positive recombinant phages identified by ELISA were used to infect E.coli TOP10 for the production of soluble Fab antibodies. Results: A schistosoma japonicum human Fab phage-display library consisting of 4.3 × 106 clones was successfully constructed. The sequencing results demonstrated that it was a human Fab gene library. After five rounds of panning, sixty randomly selected clones from the enriched phage library were tested with ELISA, and four clones(A3,B6,C4,C17) were found to bind to IMS (Ab1) but not to SEA and SWAP. The clone B6 was expressed and identified by SDS-PAGE. Sequencing was also carried out. Conclusion:A schistosoma japonicum human Fab phage-display library could be successfully constructed, from which anti-idiotypic antibody (B6) of schistosoma japonicum is obtained. And it is useful for further study on anti-idiotypic antibody vaccine development of schistosoma japonicum.

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岳国峰,张慧林,焦永军,朱 进,冯振卿,管晓虹.人源日本血吸虫Fab抗体库的构建及抗独特型抗体的筛选、鉴定[J].南京医科大学学报(自然科学版),2006,(11):997-1000

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  • 收稿日期:2006-04-09
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