前列腺特异膜抗原基因的克隆和真核表达  

Cloning and expression of human prostate-specific membrane antigen

作　　者：戴淑琴[1] 孙秀英[2] 王军业[3] 曹开源[4] 何丽容[1] 徐霖[4] 袁广卿[4] 

机构地区：[1]中山大学肿瘤防治中心检验科,广州510060 [2]山东省临沂市人民医院检验科 [3]中山大学肿瘤防治中心胸科 [4]中山大学中山医学院检验系

出　　处：《中国肿瘤临床与康复》2005年第4期289-292,共4页Chinese Journal of Clinical Oncology and Rehabilitation

基　　金：广东省自然科学基金项目(No.021907)

摘　　要：目的克隆前列腺特异膜抗原(PSMA)基因的编码区序列,并进行真核表达。方法用RT-PCR方法扩增前列腺癌组织标本中的PSMA cDNA序列,并将其克隆至真核表达载体pcDNA3.0。用脂质体转染法,把pcDNA3.0-PSMA转染哺乳动物细胞,鉴定PSMA蛋白的表达。结果序列测定结果表明,两条引物之间的片断长度为2279 bp,与预期长度一致。将克隆的编码区序列与Genebank的PSMA序列进行Blast对比分析,同源性为99.7%;间接免疫荧光法显示表达的蛋白质为膜蛋白,免疫印迹表明表达蛋白质的分子量为100 000。结论利用RT-PCR技术成功扩增了PSMA编码区序列,经序列分析显示扩增片断的序列正确。构建了PSMA真核表达载体,建立了PSMA稳定表达的细胞株。经免疫分析,证实了真核细胞内表达的PSMA为分子量正确的膜蛋白,且具有良好的抗原性。所获得的PSMA真核表达系统为进一步研究PSMA基因修饰的DCs疫苗对前列腺癌的治疗提供了物质基础。Objective To clone cDNA of human prostate-specific membrane antigen（PSMA）, construct pcDNA3-PSMA eukaryotic expression vector, and then identify the expression of PSMA protein. Methods Total RNA was extracted from prostate cancer tissues. A pair of primers according M99487 in Genebank was designed and the PSMA cDNA fragment was obtained by reverse transcritionase-polymerase chain reaction. The PCR products were then cloned into empty plasmid pcDNA3. Further sequence analysis was carried out, The recombinant plasmids pcDNA3.0-PSMA were transfected into PC-3 ceils. In G418 selective medium （400μg/ml）, two positive clones expressing PSMA mRNA, named D4 and B3 respectively, were identified by RT-PCR. Using rabbit anti-human PSMA monoclonal antibody, we identified the location of expressed PSMA protein by indirected immunofluoreseence analysis. To verified the molecular weight of expressed PSMA protein, Western blotting analysis of total cell extracts was carried out using chemiluminescence Western blotting kit. Results Sequence analysis confirmed the homogeneity between cloned PSMA cDNA and M99487 was 99.7%. After transfection, two cell clones expressing PSMA mRNA were identified by RT-PCR. The indirect immunofluoreseence assay confirmed that expressed PSMA protein was located on membrane and had good immunoreactivity. Western blotting analysis showed the molecular weight of expressed PSMA was 100 000, as was PSMA protein from LNCaP cells. Condusions We have obtained the PSMA cDNA from prostate cancer tissues. The pcDNA3.0-PSMA eukaryotic expression vector was constructed and the target PSMA protein which had immunoreactivity expressed on cell membrane. The molecular weight of expressed PSMA was 100 000, the right molecular weight as we expected. Our research layed foundation for treatment of prostate neoplasms by DC vaccines transfecte with PSMA.

关 键 词：前列腺肿瘤 前列腺特异膜抗原 克隆 分子 真核表达 

分 类 号：R737.25[医药卫生—肿瘤] R730.45[医药卫生—临床医学]