大容量天然人源Fab抗体库的构建及日本血吸虫抗独特型抗体的筛选、鉴定

Screening and identification of human Fab antibody against idiotypic antibody of schistosoma japonicum from large naive phage-display library

目的:构建大容量天然人源Fab抗体库,筛选全人源日本血吸虫抗独特型抗体并进行初步鉴定。方法:采集15位健康成人的骨髓淋巴细胞,构建天然人源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2×109大容量天然人源Fab抗体库,经核苷酸序列分析,证实插入片段为Fab基因片断。经过6轮筛选后,从富集的次级抗体库中随机挑取80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(C12、C19、D1、D2),其中C12经SDS-PAGE电泳显示插入片断正确。结论:成功构建了大容量天然人源Fab抗体库,并从中获得人源日本血吸虫抗独特型抗体C12,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。

Objective：To obtain the completely humanized Fab antibody against idiotypic antibody of schistosoma japonieum from large naive phage-display library. Methods： A naive library of phage-display human Fab library was established, genes of which were derived from bone marrow lymphocytes of 15 healthy donors. Soluble egg antigen（SEA） and soluble worm antigen preparations （SWAP） of sehistosoma japonicum immune mouse serum（IgG） were used to screen Fab antibody. The positive recombinant phages identified by ELISA were used to infect E.coli TOP10 for the production of soluble Fab antibodies. Results：A large naive human Fab phage-dis- play library consisting of 2×10^9 clones was successfully constructed. The sequencing result demonstrated that it was a human Fab gene. After 6 rounds of panning, eighty randomly selected clones from the enriched phage library were tested with ELISA. Four clones （C12,C19,D1 ,D2） were found to bind to IMS （Abl） but not to SEA and SWAP. C12 was expressed and identified by SDS- PAGE. Sequencing was also carried out. Conclusion：A human Fab large naive phage-display library might be successfully constructed, from which anti-idiotypie antibody （C12） of schistosoma japonicum could be obtained. And it may be useful to study on anti-idiotypic antibody vaccine of schistosoma japonicum.