肾癌特异性配体多肽ZT-2筛选及新标志物的研究

目的 寻找肾癌早期诊断和靶向治疗的标志物. 方法 以肾癌细胞株A498为靶细胞,正常肾细胞株HK-2为吸附细胞,37℃下对噬菌体十二肽库进行4轮减性筛选.挑取单克隆采用ELISA方法 初步鉴定噬菌体克隆亲和力;阳性克隆提取质粒,测序后合成多肽,鉴定多肽的亲和力及特异性.结果 经ELISA初步鉴定,随机挑选20个单克隆中,2号多肽对A498细胞亲和力最高(A_(ZT-2)/A_(对照)比值为3.15),命名为Phage-ZT-2.细胞免疫荧光试验证明,合成的相应多肽FITC-ZT-2对A498细胞具有特异性结合.利用荧光标记的ZT-2检测肾癌组织芯片,肾癌组织ZT-2的荧光吸光度(A)值为0.453±0.123,正常组织及非肾癌炎性组织为0.148±0.075,2组间比较差异有统计学意义(t=2.776,P<0.01).结论 成功筛选出与肾癌细胞特异性结合的多肽ZT-2,为肾癌的早期诊断和靶向治疗提供了实验依据.

Screening and identification of renal cancer specific peptide from a phage displaypeptide library

Objective To screen and identify the novel markers for renal cell carcinoma. Methods The renal cancer A498 cell line was used as the antigen and human normal renal cell line HK-2 was used as control for subtraction biopanning from a phage display peptide library at 37 ℃ The positive and specific binding clones were identified by cell-based ELISA and immunocytochemical staining, and the identified clones were sequenced. Thus the amino acid sequence was deduced and the peptide was synthesized. The peptide was identified by immunofluorescence. Results Through a cell-based ELISA, immunocytochemical staining, and immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specially bind to the A498. The affinity binding to A498 was the highest (A_(ZT-2)/A_(control) = 3. 15) among the peptides assayed. The optical density of ZT-2 in renal cancer tissue (0. 453±0. 123) was significantly higher than that in normal and inflammatory renal tissue (0. 148±0. 075)(P<0. 01). Conclusions A peptide ZT-2 which is specific binding to renal cancer cell line A498 had been selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of renal cancer or targeted drug delivery in chemotherapy.