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目的:通过错配PCR技术提高1株人源化抗hIL17A单链抗体(scFv)的亲和力,为进一步开发治疗类风湿性关节炎(RA)的人源化抗体药物奠定基础。方法:本研究采用错配PCR和重叠PCR的方法,对抗体的重链、轻链可变区分别随机引入突变,并将细菌内膜展示技术和流式细胞技术(FCM)相结合进行高通量筛选,获得高亲和力突变株。由于hIL17A能刺激HeLa细胞产生白细胞介素IL-6和IL-8,因此scFv突变株经表达纯化后,利用real-time PCR技术在mRNA转录水平上检测其阻断hIL17A刺激HeLa细胞产生IL-6和IL-8的能力。结果:筛选出5株突变株,经流式细胞仪检测其中有3株亲和力与亲本相比有很大程度的提高,2株与亲本相当,而且所有突变株均保留了亲本与hIL17A的结合能力,经real-time PCR检测证明这5株突变株均有阻断hIL17A刺激HeLa细胞产生IL-6和IL-8的能力,突变株的亲和力与中和效果成正比。结论:错配PCR技术是抗体体外亲和力提高的一种可行性方法,在保证与抗原结合能力的基础上提高了其亲和力和中和活性。该实验结果为RA的靶向治疗提供了候选药物,而且也为抗体的体外亲和力的提高提供了参考方法。
OBJECTIVE: To improve the affinity of a humanized anti-hIL17A single chain antibody (scFv) by mismatched PCR and lay the foundation for the further development of humanized anti-RA drugs for rheumatoid arthritis (RA). Methods: Mismatch PCR and overlap PCR were used to introduce mutants into the variable regions of the heavy chain and the light chain of the antibody respectively. The combination of bacterial intima display technique and flow cytometry (FCM) Amount of screening, access to high affinity mutant strains. Since hIL17A can stimulate IL-6 and IL-8 production in HeLa cells, the scFv mutant was purified by expression and then detected by real-time PCR at the transcriptional level of hIL17A to stimulate HeLa cells to produce IL-6 And IL-8 capacity. Results: Five mutant strains were screened. Among them, three of them were greatly improved by flow cytometry compared with their parents. Two of them were similar to their parents, and all the mutants retained their binding ability to hIL17A The results of real-time PCR showed that these five mutant strains could block the ability of hIL17A to stimulate the production of IL-6 and IL-8 in HeLa cells. The affinity of the mutants was proportional to the neutralizing effect. Conclusion: The mismatch PCR technique is a feasible method to improve the affinity of antibody in vitro, which enhances its affinity and neutralization activity on the basis of ensuring its binding ability with antigen. This experimental result provides a candidate drug for the targeted therapy of RA, and also provides a reference method for improving the affinity of the antibody in vitro.