抗克伦特罗单链抗体噬菌体展示文库的构建初步筛选及鉴定

Construction, Screening and Identification of scFv Phage Display Libraries against Clenbuterol

构建抗克伦特罗(CBL)单链抗体噬菌体展示文库(scFv phage displaylibraries),从中筛选CBL特异性噬菌体单链抗体(phage scFv).提取经CBL免疫的小鼠的脾脏总RNA,RT-PCR扩增全套抗体轻链可变区(VL)及重链可变区(VH)基因,重叠延伸法将VL、VH片段拼接为单链抗体(scFv)基因片段.将scFv克隆到噬菌粒载体pCANTAB5E中,转化感受态大肠杆菌TG1,用辅助噬菌体M13KO7进行超感染,构建噬菌体单链抗体库.对抗体库进行亲合富集后,ELISA法筛选阳性克隆.扩增出抗CBL的VL、VH基因片段并拼接为全长的scFv,抗体库库容约为1.6×104,经4轮"吸附-洗脱-扩增"的富集,ELISA筛选到6个具有CBL结合活性的phage-scFv.成功构建抗CBL单链抗体噬菌体展示库,并初步筛选到具有CBL结合活性的噬菌体scFv,特异性好,为进一步大量表达CBL单链抗体奠定了基础.

To construct single chain Fv （scFv） phage display libraries and screen specific phage scFv directed against clenbuterol. The total RNA was extracted from spleen of CBL-hyperimmunized mouse. The variable heavy （VH） and light （VL） genes were amplified by RT-PCR and scFv was assembled through overlapping PCR. The scFv fragments were cloned into the phagemid vector pCANTAB5E, then transformed into competent E. coli TG1 cells. After being rescued by helper phage M13KO7, scFv phage display libraries were constructed. The specified recombinant phages were enriched through affinity panning and the CBL-positive phage scFv clones were screened and identified by ELISA. The VH and VL genes were amplified and assembled to scFv fragment successfully. The size of antibody libraries is about 1.6×10^4. After 4 round of enriched procedures, ELISA screened 6 CBL-positive phage scFv clones. The scFv phage display libraries directed against CBL were constructed successfully and specificity phage scFv against CBL were screened. It is a good start for the next step to express the scFv solubly.