巨型引物PCR法对抗CD3改型单链抗体的定点诱变

Site-directed Mutagenesis of Anti-CD3 Single-chain Fv Fragment(ScFv) Using Two Side Megaprimer PCR Method

亲和力是影响改型单链抗体应用于临床的重要因素之一 .利用巨型引物PCR定点诱变方法 ,设计并化学合成出两组含多个突变位点的简并引物 ,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段 ,即巨型引物 ,将其经琼脂糖凝胶电泳分离纯化后 ,作为 3′和 5′的两端引物应用于第二轮PCR反应中 .通过改变标准PCR反应条件 ,调整引物与模板的浓度 ,扩增出特异性较强的目的DNA条带 .PCR产物经回收后 ,进行DNA测序 .

In order to improve the affinity of anti-CD3 ScFv, one site-directed mutagenesis method using 'megaprimer' PCR was designed. Adopting the mutagenic primer in the first round of PCR, mutation DNA fragments of 180 bp and 160 bp were amplified, respectively. Using these fragments as 'megaprimers' in the second round of PCR, specific full-length DNA by changing the PCR program and adjusting the concentration of the primer and template were obtained. The sequencing results showed that the mutant pool of anti-CD3 ScFv had been yielded successfully.