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目的构建肾癌G250抗原基因编码区序列的真核表达载体并进行序列测定。方法从肾癌组织中提取总RNA,采用RT-PCR技术扩增肾癌G250抗原的基因编码区序列,将PCR片段定向克隆至真核表达载体pcDNA3.0,并进行序列测定。结果肾癌组织RT-PCR扩增后,均产生特异性条带,位置在1000 bp和1600 bp之间,与预定相符。pcDNA3.0-G250分别用H indⅢ、XhoⅠ双酶切后产生2条带,均与预定位置相符;抗原基因编码区序列与Genbank登录号BC014950文献报道的序列同源性为100%,目的基因与载体正确连接。结论成功克隆了肾癌G250抗原的基因编码区序列,并构建了其真核表达载体pcDNA3.0-G250,为核素标记的G250单克隆抗体在诊断和治疗的应用及G250基因修饰的树突状细胞疫苗的肾癌生物治疗提供了依据。
Objective To construct the eukaryotic expression vector of the coding region of G250 antigen of renal cell carcinoma and to determine its sequence. Methods Total RNA was extracted from renal carcinoma tissues. The coding region of G250 antigen of renal cell carcinoma was amplified by RT-PCR. The PCR fragment was cloned into eukaryotic expression vector pcDNA3.0 and sequenced. Results After RT-PCR amplification of renal cell carcinoma, specific bands were produced, located between 1000 bp and 1600 bp, in agreement with the prediction. pcDNA3.0-G250 were digested with H indⅢ and XhoⅠ, respectively, which were consistent with the expected positions. The sequence of the coding region of the antigen gene was 100% identical to that reported in the GenBank accession number BC014950, The carrier is properly connected. Conclusion The coding sequence of G250 antigen of renal cell carcinoma was successfully cloned and its eukaryotic expression vector pcDNA3.0-G250 was constructed. The application of radionuclide labeled G250 monoclonal antibody in the diagnosis and treatment and G250 gene modified dendrites Cell-based vaccine provides a basis for renal cancer biotherapy.