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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 the coding region of prostate specific membrane antigen (PSMA) gene and perform eukaryotic expression. Methods PSMA cDNA sequence of prostate cancer tissue was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.0. Liposome transfection method, the pcDNA3.0-PSMA transfected mammalian cells, to identify the expression of PSMA protein. Results The sequencing results showed that the length of the fragment between the two primers was 2279 bp, consistent with the expected length. Blast analysis of the cloned coding region sequence and Genebank’s PSMA sequence revealed that the expressed protein was a membrane protein with a homology of 99.7%. The molecular weight of the expressed protein was 100 000 by indirect immunofluorescence. Conclusion The sequence of PSMA coding region was successfully amplified by RT-PCR. Sequence analysis showed that the sequence of amplified fragment was correct. The PSMA eukaryotic expression vector was constructed and a cell line stably expressing PSMA was established. Immunoassay confirmed that PSMA expressed in eukaryotic cells was the correct molecular weight of membrane protein and had good antigenicity. The obtained PSMA eukaryotic expression system provides a material basis for the further study of PSMA gene modified DCs vaccine for the treatment of prostate cancer.