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人前列腺特异性膜抗原cDNA的克隆、原核表达、鉴定和抗原初步纯化 被引量:6

Prokaryotic expression,identification,primary purification of human prostate specific membrane antigen
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摘要 目的 克隆中国人前列腺特异性膜抗原 (PSMA)cDNA全长 ,构建PSMA原核表达重组子。诱导PSMA原核表达 ,并对表达产物进行初步纯化。 方法 从前列腺癌患者组织中提取总RNA ,利用PSMAcDNA中的EcoRⅠ位点 ,将PSMA分为两段分别作RT PCR ,分别连接到pET 30a中 ,从而得到完整PSMAcDNA的原核表达重组子pET 30a PSMA ,并进行酶切鉴定与序列测定。确定序列正确后 ,利用IPTG诱导重组子表达 ,采用Ni NTAAgarose螯合层析柱对表达产物初步纯化 ,SDS PAGE和Western Blotting对表达产物和纯化产物进行鉴定。 结果 成功克隆到序列完全正确的人源PSMAcDNA ,诱导表达并初步纯化出PSMA蛋白 ,该蛋白具有较好的抗原性和特异性。 Objective To describe the cloning of cDNA of prostate specific membrane antigen (PSMA),construction of the recon of PSMA,induction of the expression of 6 × His PSMA fusion protein from PSMS recon,and purification of the expression products. Methods The total RNA was extracted from prostate cancer tissues.The site of EcoR I lying in the middle of PSMA’s cDNA was used to perform the reverse transcriptase and polymerase chain reaction of PSMA in two different parts,which were then linked into the carrier of pEt 30(a),thus the full cDNA of PSMA and the pronucleus expression carrier pET 30(a) PSMA were obtained.The pET30a (+) PSMA recon was transformed into the Escherichia coli BL 21 and the engineering bacteria expressing protein PSMA was gotten. With the induction of IPTG, the recon was expressed. The expression products were purified with Ni NTA Agarose resin, then the purer protein PSMA was obtained. The antigenicity and specificity of the expressed PSMA were evaluated with Western blotting and SDS PAGE. Results The wholly right base sequence of PSMA’s cDNA was successfully cloned.PSMA protein was expressed and purified.This protein had better antigenicity and specificity. Conclusions This study provides experimental basis for the function study of PSMA and scanning of the phage antibody library.
出处 《中华泌尿外科杂志》 CAS CSCD 北大核心 2004年第8期533-536,共4页 Chinese Journal of Urology
基金 广东省自然科学基金 ( 0 0 13 5 6) 中山大学第二附属医院重点项目基金
关键词 前列腺 特异性 膜抗原 CDNA 克隆 原核表达 抗原 PSMA 肿瘤 Prostatic neoplasms Carcinoma Prostate specific membrane antigen
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参考文献5

  • 1Israeli RS,Powell CT,Fair WR,et al.Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen.Cancer Res,1993,53,227-230.
  • 2Ruth E,Alexis R,Joseph T.Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristic of a neuropeptidase.Proc Natl Acad Sci,1996,93:749-753.
  • 3Gregorakis AK,Holmes EH,Murphy GP.Prostate-specific membrane antigen:current and future utility.Semin Urol Oncol,1998,16:2-12.
  • 4Bostwich DG,Pacelli A,Blute M,et al.Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma:a study of 184 cases.Cancer,1998,82:2256-2261.
  • 5Hengen,Paul N.Methods and reagents-purification of His-tag fusion protein from Escherichia coli.Trends Bio Sci,1995,20:285-286.

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