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

Screening and identification of human phage displayed sc Fv antibody against ATPbinding cassette sub-family G member 2 of lung adenocarcinoma

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sc Fv,克隆入噬菌体表达载体p CANTAB5E,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。Western blot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。Western blot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sc Fv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。

Object ve To construct a phage display library and screen phage-displayed single-chain variable fragment （scFv） antibody against ATP-binding cassette sub-family G member 2 （ABCG2） of lung adenocarcinoma. Methods Total RNA of positive lymph nodes adjacent to lung adenocarcinoma was extracted. Variable regions of heavy （VH） and light chain （VL） were amplified by PCR. ScFv gene was synthesized by SOE-PCR, and then linked to a pCANTABSE phagemid vector to finally create a phage displayed scFv library. Six rounds of biopanning against A549 cells and ABCG2 antigen were performed. The properties of the product were identified by Western blotting, and its specificity was detected by ELISA and immunocytochemical assay. CCK8 assay was used to detect its effect on cisplatinsensitization. Results A phage displayed scFv antibody against ABCG2 of lung cancer was successfully constructed. The obtained scFv antibody had an apparent molecular weight of 34 ~ 103 and significantly down-regulated the expression of ABCG2 protein in A549 cells. Besides, it demonstrated a relative specific affinity to A549 cells and ABCG2 antigen with a positive recognition rate of 75%. Furthermore, it increased the cisplatin sensitivity in A549 cells with distinctly reduced ICs0 values. Conclusion A phage displayed anti-ABCG2 scFv of lung cancer issuccessfully constructed, and through it, scFv antibody is screen out which can specifically bind to single- chain antibodies. This study found a strong basis for subsequent body visualization research and lung cancer stem-like cells targeting ABCG2.