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目的 建立真核表达重组体 pc DNA- BL C的稳定转染细胞株 ,为研究趋化因子 BL C的抗肿瘤免疫作用机制奠定基础。方法 以小鼠脾脏组织总 RNA为模板 ,通过 RT- PCR扩增 BL C全长 c DNA,并将扩增的 c DNA片段插入 pc DNA3.1(+)真核表达质粒 ,构建重组质粒 pc DNA- BL C,重组子经限制性内切酶酶切分析及测序鉴定后 ,用脂质体转染技术导入 Colon2 6细胞 ,经 G4 18筛选建立稳定转染细胞株。用免疫印迹法 (Western Blot)鉴定转染细胞表达的蛋白质 ,证实 BL C存在。用趋化实验证实转染细胞株表达的 BL C有生物学活性。结果 真核表达重组体 pc DNA- BL C构建成功 ,pc DNA- BL C稳定转染细胞株表达有生物学活性的 BL C。结论 成功建立了 BL C的稳定转染细胞株 ,为研究 BL C的抗肿瘤免疫作用机制继而发展肿瘤疫苗奠定了基础。
OBJECTIVE: To establish a stable transfected cell line expressing pcDNA-BL C by eukaryotic expression, which lays the foundation for studying the anti-tumor immune mechanism of chemokine BL C. Methods The total RNA of BL C was amplified by RT-PCR using the total RNA of spleen tissue of mice as a template. The amplified c DNA fragment was inserted into pcDNA3.1 (+) eukaryotic expression plasmid to construct recombinant plasmid pcDNA - BL C Recombinants were identified by restriction analysis and sequencing. The recombinant plasmid was transfected into Colon2 6 cells by lipofection technique. Stably transfected cell lines were selected by G418 screening. The proteins expressed in the transfected cells were identified by Western Blot, confirming the presence of BLc. The chemotactic experiment was used to confirm the biological activity of BL C expressed in the transfected cell lines. Results The eukaryotic expression recombinant pcDNA-BL C was successfully constructed and the pcDNA-BL C stably transfected cell line expressed the biologically active BL C. Conclusion The stable transfected cell line of BL C was successfully established, which laid the foundation for the study of anti-tumor immune mechanism of BL C and the development of tumor vaccine.