摘要
目的:克隆人肾细胞癌特异性抗原G250基因,使其能够在真核细胞中表达并锚定修饰于细胞膜上。方法:提取人肾透明细胞癌细胞的总RNA,用RT-PCR扩增出G250基因,插入含有人Igκ链前导信号肽、IgG-Fc和GPI锚定信号肽基因序列的真核表达载体pCI-neo-GPI中;将构建的重组质粒pCI-neo-GPI-G250转染RenCa细胞,利用RT-PCR、Western印迹和免疫荧光检测G250在细胞中的表达。结果:构建的重组质粒pCI-neo-GPI-G250经测序正确;用各种方法对筛选得到的稳定转染细胞进行检测,均可以发现G250的表达,表达部位为细胞膜。结论:克隆出人肾细胞癌特异性抗原G250基因,且G250在RenCa细胞膜上可以正常表达,为进一步研究肾癌肿瘤疫苗的免疫原性提供了必要的前期准备。
Objective： To clone the gene of G250/MN/CA Ⅸ, a specific antigen of renal cell carcinoma, and evaluate the expression of pCI-neo-GPI-G250 in the cellular membrane. Methods： The total RNA of G250 was extracted from the renal cell carcinoma cells and G250 gene was cloned by RT-PCR. Recombinant plasmid pCI-neo-GPI-G250 was constructed by inserting G250 into an eukaryotic expression vector pCI-neo-GPI that included the genes of the targeting signal peptide of IgK, IgG-Fc and GPI. And then, pCI-neo-GPI-G250 was transfected to the RenCa cell successfully. The expression of G250 was detected by RT-PCR, Western blotting and IMF. Results： The sequence of recombinant plasmid pCI-neo-GPI-G250 was consistent with that of design and the expression of G250 was detected on the cellular membrane. Conclusions：G250 gene is cloned successfully and detected its normal expression on the RenCa cellular membrane. These results have provided necessary basises to research the immunogenicity of renal cell carcinoma tumor vaccine further.
出处
《生物技术通讯》
CAS
2006年第4期496-499,共4页
Letters in Biotechnology
基金
国家高技术研究发展计划项目(2001AA217131)
