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本文将热稳定抗原(HSA)的CDNA与质粒PcDNA3连接,构建成真核表达体,通过电穿孔的方法将重组质粒导入不表达HSA的淋巴瘤细胞EL-4中,使其表达抗性基因和HSA分子,通过高剂量G418(400μg/ml)选择培养和流式细胞仪免疫荧光染色检测,并进一步通过有限稀释法克隆到高表达HSA的单细胞克隆,用含低剂量G418(200μg/ml的RP-MI1640完全培养液维持培养,以维持转染瘤细胞上的HSA的表达,从而提供活化T细胞必要的协同刺激信号,以诱导出有效的抗肿瘤免疫应答.实验观察了高表达HSA淋巴瘤细胞EL-4在体内诱导的抗癌效应及其提高肿瘤免疫原性的作用.结果表明,用丝裂霉素C灭活的HSA~+癌细胞作为瘤苗早期治疗野生型瘤细胞EL-4接种小鼠的动物模型显示
In this article, the CDNA of heat-stable antigen (HSA) was ligated with plasmid pcDNA3 to construct eukaryotic expression plasmid. The recombinant plasmid was introduced into EL-4 cells that do not express HSA by electroporation to express the resistance gene. HSA molecules were selected by high-dose G418 (400μg/ml) selection culture and flow cytometric immunofluorescence staining, and further cloned by limiting dilution into HSA-expressing single cell clones, with low doses of G418 (200μg/ml RP-MI1640 complete culture medium was maintained in culture to maintain the expression of HSA on the transfected tumor cells, thereby providing the necessary co-stimulatory signals of activated T cells to induce effective anti-tumor immune responses. Experimentally observed high expression of HSA lymphoma Anti-cancer effect induced by EL-4 cells in vivo and its role in enhancing tumor immunogenicity. The results show that HSA~+ cancer cells inactivated with mitomycin C are used as tumor vaccines for early treatment of wild-type tumor cells EL-4. Inoculated animal model display