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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
引用本文:叶传忠, 赵旭东, 张芳林, 林震, 许明, 张永康, 陈常庆,.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[J].生物工程学报,2002,18(1):35-39.
作者姓名:叶传忠  赵旭东  张芳林  林震  许明  张永康  陈常庆  
作者单位:1. 复旦大学附属中山医院泌尿外科,上海,200032
2. 中国科学院上海生物工程研究中心,上海,200042
3. 上海第二医科大学附属瑞金医院上海内分泌研究所,上海,200025
4. 福建医科大学附属协和医院泌尿外科,福州,350001
基金项目:复旦大学研究所专项基金资助项目 (U 2 )~~
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 ,该抗体可用于前列腺癌组织标本PSMA表达的检测

关 键 词:前列腺特异膜抗原膜外区    原核表达    亲和层析    抗体  
文章编号:1000-3061(2002)01-0035-05
修稿时间:2001年7月30日

Cloning and Expression of Extracellular Domain of Prostate Specific Membrane Antigen in Escherichia coli and Preparation of Polyclonal Antibody
YE Chuan-Zhong,\ ZHAO Xu-Dong \ ZHANG Fang-Lin \ LIN Zhen,XU Ming \ ZHANG Yong-Kang \ CHEN Chang-Qing.Cloning and Expression of Extracellular Domain of Prostate Specific Membrane Antigen in Escherichia coli and Preparation of Polyclonal Antibody[J].Chinese Journal of Biotechnology,2002,18(1):35-39.
Authors:YE Chuan-Zhong  \ ZHAO Xu-Dong \ ZHANG Fang-Lin \ LIN Zhen  XU Ming \ ZHANG Yong-Kang \ CHEN Chang-Qing
Institution:Department of Urology, Zhongshan Hospital, Medical Center of Fudan University, Shanghai 200032, China. chuanzhong@mycity.com.cn
Abstract: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(120 kD). BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody. The polyclonal antibody, which had a title of 1:12,800, were indicated the specificity to prostate tissue.
Keywords:extracellular domain of prostate-specific membrane antigen  expression  affinity chromatography  fusion protein  polyclonal antibody
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