抗速灭威噬菌体单链抗体库的构建、筛选及鉴定

摘要

Balb/C mice were immunized with a hapten conjugate of Metolcarb, and their spleen cells were harvested. VH and VL genes were amplified by RT-PCR. VH and VL were matched into ScFc by overlap extension PCR, and cloned into pCANTAB5E expression vector, and then transformed TG1 cells. Thus the phage single-chain Fv(ScFv) library with a size of about 7.6×105 was successfully constructed. The library was biopanned by 5 rounds of binding-elution-enrichment procedure;and 6 positive clones with high specificity for Metolcarb were obtained. The results generated could be the foundation for large-scale production of specific antibodies against Metolcarb.%以速灭威抗原免疫的小鼠为实验对象,取其脾细胞,RT-PCR扩增抗体重链可变区(VH)和轻链可变区(VL)基因,重叠延伸PCR拼接单链抗体(ScFv)基因,克隆入噬菌粒表达载体pCANTAB5E并转化大肠杆菌TGl,成功构建了库容约7,6×105的抗速灭威噬菌体ScFv库.对该文库进行了5轮"吸附-洗脱-扩增"的富集筛选,获得了6个特异性较强的抗速灭威噬菌体ScFv阳性克隆.其研究结果为速灭威特异性抗体的大量制备奠定了一定的基础.