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三磷酸腺苷结合转运蛋白G超家族成员2特异性人源肺癌噬菌体单链抗体的筛选和鉴定

, PP. 2343-2347

Keywords: 噬菌体单链抗体scFv,肺腺癌,ABCG,靶向治疗,化疗增敏

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Abstract:

目的通过噬菌体展示库技术构建人肺癌单链抗体库,并筛选出抗多药耐药蛋白ABCG2的特异性单链抗体。方法取肺腺癌患者癌旁阳性淋巴结提RNA,PCR扩增重链和轻链可变区基因VH、VL,SOE-PCR扩增单链抗体可变区基因scFv,克隆入噬菌体表达载体pCANTAB5E,制备初级抗体库。以A549细胞及ABCG2蛋白对抗体库进行6轮淘选。Westernblot检测单链抗体的可溶性表达和对ABCG2蛋白表达的影响,ELISA和ICC鉴定其特异性,CCK-8检测其顺铂增敏作用。结果成功筛选出抗ABCG2人源肺腺癌噬菌体单链抗体。Westernblot证实其可溶性表达,大小约为34×103且可明显下调ABCG2蛋白的表达。ELISA和ICC检测到该抗体对ABCG2抗原的识别率高达75%,并与A549细胞具有结合特异性。CCK8结果则显示该scFv可增加A549细胞对顺铂的敏感性,明显减小其IC50值。结论成功构建了人源肺腺癌噬菌体单链抗体库,并筛选出了能与ABCG2蛋白特异性结合的单链抗体,为后续体内可视化研究和靶向ABCG2+的肺癌干样细胞奠定基础。

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