摘要
采用表位合成多肽免疫获得编码人源抗体的基因材料,抗体轻重链可变区基因通过剪接重叠延伸(SOE)技术进行组装后,亚克隆入噬菌体粒中构建免疫文库。以重组制备的A型肉毒毒素保护性抗原为配体筛选结合子,ELISA鉴定,获得具有较高抗原结合力的噬菌体克隆,进行基因测序和家族定位分析。结果显示,表位合成多肽免疫,成功构建了库容大于108的重组抗体文库。体外3轮免疫淘筛,筛选到人源A型肉毒抗毒素克隆,基因结构分析表明,其中ScFvB17全长750bp,可编码250个氨基酸残基:VH属于VH4家族,369bp,编码123个氨基酸残基;Vκ属于κ链Ⅱ家族,336bp,编码112个氨基酸残基。基因的同源分析表明,这是一株特异的单链抗体新基因。
The genetic material encoding for human antibodies library could be obtained from immunized donors by neurotoxin epitopes peptides. V genes of H chains and κ chains were linked through a polypeptide spacer((Sergly4)3) by the splicing overlap extension(SOE) technology. Assembled 750 bp ScFv was subcloned into a phagemid pCANTAB-5E. Panning of the phage library was performed in microplates coated with recombinant toxin antigen. Phage clones were identified by ELISA and the positives were sequenced and analyzed. As a result, a human phage antibodies library was constructed with the size of over 108. After rounds of panning, phage-Ab were enriched with affinity maturation, and rare phage binders were selected. Of them, clone B17 is of high affinity and specificity to target antigen. VH of B17 is of 369 bp in length, Vκ is of 336 bp, ScFv is of 750 bp encoding 250 amino acids. Analysis results showed V-genes of B17 are new antibody Fv genes.
出处
《生物技术通讯》
CAS
2004年第5期441-443,共3页
Letters in Biotechnology
基金
国家重点基础研究发展规划("973"计划)(2002CB513205)
总后"十五"指令性课题(IL033)
