前列腺特异膜抗原在Pichia pastoris酵母中的表达及鉴定

前列腺特异膜抗原是一种具有高度前列腺特异性的糖蛋白 ,其表达的增高与肿瘤的术后复发、激素抵抗及较差的预后正相关 ,在前列腺癌的诊断和治疗中有广泛的应用前景 .从前列腺癌组织提取总RNA ,利用RT PCR技术获得PSMA基因的全长序列 ,构建了重组酵母表达载体pPIC3.5K PSMA .电击法转化Pichiapastoris酵母 ,通过表型筛选和PCR鉴定证实该基因已稳定整合入Pichiapastoris酵母基因组中 .SDS PAGE显示 ,获得的PSMA蛋白分子量约 10 0kD ,表达产物经West ern印迹证实可特异地与PSMA单克隆抗体 4G5结合 .结果表明 ,成功地获得PSMA编码的cDNA并在酵母细胞中获得表达 .

Expression and Characterization of Prostate-specific Membrane Antigen in Pichia pastoris Yeast System

Prostate specific membrance antigen(PSMA) is a highly prostate restricted glycoprotein that is elevated in prostate cancers,especially in poorly differentiated, metastatic and hormone refractory carcinomas. It is a useful prognostic biomarker and target for developing novel therapeutic strategies. PSMA cDNA was amplified from human prostate carcinoma tissue by RT PCR and sequenced. The expression vector pPIC3.5K with PSMA gene was constructed and transformed into Pichia pastoris GS115 strain by electroporation. Positive recombinant was isolated by PCR and its phenotype was determined. After methanol induction, the expressed PSMA comes up to 10% of total proteins. The molecular weights of recombinant PSMA was 100 kD.Western blot analysis showed the expressed protein presented the specific PSMA antigenicity. The PSMA protein obtained lays foundation for studying its biological actions and potential use in diagnosis of prostate neoplasms.