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目的:从噬菌体单链抗体库筛选特异性的抗人表皮生长因子(hEGF)抗体并进行活性鉴定。方法:以hEGF为抗原,对所建抗体库进行3轮“吸附-洗脱-扩增”的亲和筛选。将筛选后的单链抗体(scFv)通过ELISA和基因测序分析等方法进行鉴定;利用MTT法在体外培养的人乳腺癌细胞系MCF-7/Her-2+中检测抗体的生物结合活性。结果:在亲和筛选过程中,乳腺癌噬菌体scFv得到富集,收获率逐轮得到提高,3轮筛选之后,得到了3株高亲和力的抗hEGF抗体;ELISA检测筛选出的3种有较高活性;测序结果与人hEGF抗体基因序列高度一致;该抗体对MCF-7/Her2+乳腺癌细胞系有特异性杀伤作用。结论:成功地筛选到特异性抗hEGF的scFv抗体,为今后的研究和应用提供依据。
Objective: To screen and identify the specific anti-human epidermal growth factor (hEGF) antibodies from phage scFv library. Methods: Using hEGF as antigen, the constructed antibody library was subjected to 3 rounds of affinity screening by “adsorption-eluting-amplification”. The scFv was identified by ELISA and gene sequencing analysis. The biological activity of the antibody was detected by MTT method in human breast cancer cell line MCF-7 / Her-2 + cultured in vitro. Results: In the process of affinity screening, breast cancer phage scFv was enriched and the harvest rate was improved round by wheel. After three rounds of screening, three high affinity anti-hEGF antibodies were obtained. Three of the three screened by ELISA were higher The sequencing results were highly consistent with the sequence of human hEGF antibody. The antibody had specific killing effect on MCF-7 / Her2 + breast cancer cell line. Conclusion: The scFv antibody specific to hEGF was successfully screened, which may provide the basis for future research and application.