摘要
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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2002年第1期35-39,共5页
Chinese Journal of Biotechnology
基金
复旦大学研究所专项基金资助项目 (U 2 )~~
关键词
人前列腺特异膜抗原
膜外区
抗体
制备
原核表达
亲和层析
基因克隆
前列腺癌
extracellular domain of prostate-specific membrane antigen, expression, affinity chromatography, fusion protein, polyclonal antibody