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目的:构建小鼠GM-CSF基因的高效真核表达载体,筛选导入该载体后高水平表达GM-CSF的小鼠淋巴瘤细胞系RMA,并探讨转GM-CSF基因瘤苗治疗小鼠T淋巴细胞瘤的方法。方法:PCR扩增小鼠CM-CSF cDNA3’端770bp的片段,将其插入真核表达载体pcDNA3;用电穿孔法将构建的载体导入小鼠淋巴瘤细胞系RMA,有限稀释法制备单个细胞克隆,经RT-PCR、骨髓祖细胞增殖实验和集落形成实验筛选相对高表达GM-CSF的RMA克隆,该克隆细胞经丝裂霉素灭活后免疫小鼠以诱导其产生抵抗RMA肿瘤细胞再攻击的能力。结果:构建的重组质粒含有预期片段,插入方向正确,核酸序列无误;且获得了高表达GM-CSF的RMA克隆,将其用丝裂霉素灭活免疫小鼠后使它们产生了抗肿瘤免疫保护力。结论:转GM-CSF基因瘤苗可能作为有效的抗T淋巴细胞瘤瘤苗。
OBJECTIVE: To construct an eukaryotic expression vector for murine GM-CSF gene and to screen the mouse lymphoma cell line RMA expressing high levels of GM-CSF after it was introduced into the vector. Cell tumor method. METHODS: A 770 bp fragment of mouse CM-CSF cDNA was amplified by PCR and inserted into the eukaryotic expression vector pcDNA3. The constructed vector was introduced into the mouse lymphoma cell line RMA by electroporation, and a single cell clone was prepared by limiting dilution , RMA clones with relatively high GM-CSF expression were screened by RT-PCR, myeloid progenitor cell proliferation assay and colony formation assay. The cloned cells were inoculated with mitomycin and then inoculated to induce anti-RMA tumor cells to re-attack Ability. Results: The constructed recombinant plasmids contained the expected fragments with correct insertion direction and correct nucleic acid sequence. The RMA clones highly expressing GM-CSF were obtained, which were then immunized with mitomycin to inactivate mice to produce anti-tumor immunity Protection. Conclusion: Transfection of GM-CSF gene vaccine may be an effective anti-T lymphoma tumor vaccine.