人前列腺特异膜抗原膜外区基因的克隆表达及抗体的制备

Cloning and Expression of Extracellular Domain of Prostate Specific Membrane Antigen in Escherichia coli and Preparation of Polyclonal Antibody

利用RT PCR技术 ,从前列腺癌组织总RNA中扩增人前列腺特异膜抗原 (PSMA)基因编码区序列 ,克隆至pcDNA3.1载体 ,以此为模板再次PCR扩增出PSMA膜外区cDNA(edPSMA) ,序列测定表明克隆获得的PSMA及edPSMA与基因库所登录的序列相一致。构建原核表达质粒pMAL c2x edPSMA ,经IPTG诱导表达的MBP edPSMA融合蛋白分子量约 12 0kD ,Westernblot证实表达产物可特异地与PSMA单克隆抗体 4G5结合。用直链淀粉琼脂糖凝胶 (Amyloseresin)亲和层析纯化蛋白质可得到电泳均一的融合蛋白 ,免疫BALB C小鼠制备多抗 ,获得效价为 1∶12 80 0的多克隆抗体 。

Human Prostate Specific Membrane Antigen(PSMA) cDNA was amplified using total RNA extracted from prostate carcinoma tissue by RT-PCR.The cDNA fragment of extracellular domain of PSMA(edPSMA) gene was amplified by PCR and cloned into expression vector pMAL-c2x.Sequence analysis of both PSMA and edPSMA revealed identity to the GenBank reported. The edPSMA was expressed in E.coli as part of a fusion protein with MBP as the induction of IPTG. Western blot analysis showed the recombinant protein could react with PSMA monocloned antibodies 4G5. MBP-edPSMA fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE(120kD). BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody.The polyclonal antibody,which had a title of 1∶12800,were indicated the specificity to prostate tissue.