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目的:提高抗TNFα单链抗体(scFv)的亲和力。方法:应用错配PCR技术在抗TNFαscFv基因中引入随机突变,再通过DNA交换(shuffling)使引入的点突变进一步重排组合,构建突变噬菌体抗体库。采用硫氰酸盐洗脱法对抗体库进行淘筛。挑选活性得到改良的克隆,用斑点ELISA法及硫氰酸盐洗脱ELISA法评估其亲和力的改善。结果:对PCR错配的突变库筛选未得到亲和力明显改善的抗体变种,经DNA交换进一步构建变种库后,筛选获得1个亲和力提高的克隆,其相对亲和指数为1.37mol/L,较亲本抗体的相对亲和指数0.48mol/L有明显提高。结论:通过错配PCR和DNA交换引入随机突变构建抗体库,可有效地提高scFv的亲和力。
Objective: To improve the affinity of anti-TNFα single chain antibody (scFv). Methods: Random mutation was introduced into anti-TNFαscFv gene by mismatched PCR technique, and the introduced point mutations were further rearranged by DNA shuffling to construct mutant phage antibody library. Antibody pools were panned using thiocyanate elution. Clones with improved activity were selected and their affinity improvements assessed by dot ELISA and thiocyanate elution ELISA. Results: Mutants of PCR mismatch library were screened for antibody variants with no significant improvement of affinity. After further construction of the mutant library by DNA exchange, one clone with higher affinity was screened with a relative affinity index of 1.37 mol / L, Antibody relative affinity index 0.48mol / L significantly improved. Conclusion: It is effective to improve the affinity of scFv by introducing a random mutation into the antibody library through mismatched PCR and DNA exchange.