【摘要】 目的构建大容量、多样性的人源核糖体展示(Ribosome display,RD)单链抗体(Singe chain Fv segment,scFv)库。方法收集20名健康献血者的新鲜外周血,分离淋巴细胞,提取细胞总RNA;设计兼并引物,利用RT-PCR分别扩增人抗体的VH-linker、Vκ、Vλ及Cκ基因;采用重叠PCR(SOE-PCR)技术,构建VH-linker-Vκ和VH-linker-Vλ-Cκ单链抗体库;将VH-linker-Vκ和VH-linker-Vλ-Cκ等量混合后,与pMD19-T载体连接,并转化感受态E.coli DH5α,对所构建的scFv库进行菌落PCR和测序分析。结果构建的核糖体展示单链抗体库重组率较高,转化产物经菌落PCR鉴定,均可见约1 000 bp的scFv基因片段,库容量为2.06×1013。经分析该抗体库单链抗体序列均完整,内部无终止密码子,可变区序列无一重复,多样性良好,均具有完整的体外核糖体展示框架。结论已成功构建了大容量、多样性好的人源核糖体展示单链抗体库,为进一步筛选高特异性、高亲和力的人源抗体奠定了基础。更多还原

【Abstract】 Objective To construct high-capacity human ribosome display single chain Fv(scFv) library with diversity.Methods Fresh peripheral blood samples were collected from 20 healthy donors,from which lymphocytes were isolated for extraction of total RNA.By using the designed degenerate primer,the whole human antibody variable region of heavy chain(VH-linker),variable region of light chain including types κ and λ(Vκ and Vλ) and consist region of type κ light chain(Cκ) for space were amplified by RT-PCR,based on which the scFv libraries of VH-linker-Vκ and VH-linker-Vλ-Cκ were constructed by splicing by overlap extension PCR(SOE-PCR).The VH-linker-Vκ and VH-linker-Vλ-Cκ at equal volumes were mixed and linked to vector pMD19-T,and the constructed recombinant plasmid was transformed to competent E.coli DH5α,and the positive clones were identified by PCR and sequencing.Results The constructed ribosome display scFv library,with a capacity of 2.06 × 1013,showed high recombination rate.PCR showed scFv gene fragment at a length of about 1 000 bp in transformants.All the single chain antibody sequences in the library were intact,with intact in vitro ribosome display frame,in which no termination codons or repeat variable region sequences were observed,indicating a high diversity.Conclusion A human ribosome display single chain Fv library with high capacity and diversity was successfully constructed.更多还原