前列腺干细胞抗原(PSCA)的表达及其特异结合肽的筛选

Expression of Prostate Stem Cell Antigen (PSCA) and Selection of its Specific Binding Peptide

通过反转录 PCR从人前列腺癌细胞中克隆了前列腺干细胞抗原 (PSCA)基因 ,在大肠杆菌中利用pQE30载体对截断型PSCA基因进行了可溶性表达。蛋白纯化后 ,利用噬菌体随机展示 12肽库筛选了PSCA蛋白的特异结合肽 ,通过与EGFP蛋白的耦联表达验证了结合肽的特异性。此特异结合肽的获得 。

Prostate stem cell antigen (PSCA), a homologue of the Ly 6/Thy 1 family of cell surface antigen, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. To obtain the specific peptide binding with PSCA for targeted immunotherapy, PSCA gene was obtained by RT PCR from human prostate cancer cell line DU145 and the transcated PSCA (tPSCA) gene was cloned into vector pQE30 for soluble expression in E.coli . The identity of recombinant tPSCA was confirmed through ELISA and western blot by use of anti PSCA monoclonal antibody. Then the 12 peptide phage display library was screened with the purified tPSCA protein for its specific binding peptide through 3 rounds panning. For identifying the peptide's specificity, the peptide was coupled with EGFP(enhanced green fluorecent protein) by recombinant DNA technology and the recombinant coupled protein was termed 11 EGFP. The binding specificity with tPSCA of 11 EGFP was further confirmed by ELISA and competitive inhibition experiment. Flow cytometry demonstrated its binding specificity with cell line DU145 In conclusion, a 12 amino acid peptide which could bind with PSCA specifically was found and it may be a potential tool for targeted immunotherapy of prostate carcinoma.