人CD80-IgG1Fc融合蛋白的表达、鉴定及其诱导的抗肿瘤效应

发布时间：2018-05-28 17:53

本文选题：CD80 + IgG1　；参考：《福建医科大学》2011年硕士论文

【摘要】：目的:构建人CD80-IgG1Fc融合蛋白的真核表达载体,并转染CHO细胞,建立CHO融合蛋白稳定表达株;探讨经融合蛋白修饰的肝癌细胞H22对淋巴细胞抗肿瘤免疫的影响,及其对肝癌细胞H22皮下移植型荷瘤小鼠肿瘤生长的影响。方法:通过RT-PCR从人外周血单个核细胞中扩增CD80膜外段及IgG1Fc基因,构建真核表达载体CD80-IgG1Fc/pcDNA3.1(+),将其转染CHO细胞株并筛选。通过RT-PCR、Western blot及ELISA鉴定融合基因的表达;将融合蛋白修饰小鼠肝癌细胞株H22,流式细胞术检测细胞膜上CD80的表达,以MTT比色法、乳酸脱氢酶释放试验检测经修饰的H22细胞对脾淋巴细胞增殖及其细胞毒性的影响。建立肝癌细胞H22皮下移植型荷瘤小鼠模型后,接种经融合蛋白修饰的H22细胞,观察其对肿瘤生长的影响。结果:成功构建了真核表达载体CD80-IgG1Fc/pcDNA3.1(+),并获得稳定表达CD80-IgG1Fc融合蛋白的重组CHO细胞株。针对融合蛋白修饰后的H22细胞,脾淋巴细胞增殖较对照组增强,CTL的细胞毒活性增高。荷瘤小鼠接种经融合蛋白修饰的H22细胞后,皮下肿瘤体积在第12天小于对照组。结论:CD80 -IgG1Fc融合蛋白能在CHO细胞中稳定表达;用CD80 -IgG1Fc融合蛋白修饰的H22细胞能促进淋巴细胞活化,诱导特异性抗肿瘤免疫效应,并对体内肿瘤生长有抑制作用。
[Abstract]:Aim: to construct the eukaryotic expression vector of human CD80-IgG1Fc fusion protein and transfect it into CHO cells to establish a stable expression strain of CHO fusion protein, and to investigate the effect of H22 modified by fusion protein on the anti-tumor immunity of lymphocytes. And its effect on tumor growth of H 22 subcutaneous transplanted tumor bearing mice. Methods: the extracellular segment of CD80 and IgG1Fc gene were amplified from human peripheral blood mononuclear cells by RT-PCR, and the eukaryotic expression vector CD80-IgG1Fcr / pcDNA3.1 was constructed. The vector was transfected into CHO cell line and screened. The expression of the fusion gene was identified by RT-PCR Western blot and ELISA, and the expression of CD80 on the cell membrane was detected by flow cytometry, and the expression of CD80 was detected by MTT colorimetric assay, and the fusion protein was modified into mouse hepatoma cell line H22. The effects of modified H 22 cells on the proliferation and cytotoxicity of splenic lymphocytes were detected by lactate dehydrogenase release assay. H22 cells were inoculated with H22 cells modified by fusion protein to observe the effect of H22 cells on tumor growth. Results: the eukaryotic expression vector CD80-IgG1Fcr / pcDNA3.1 was successfully constructed, and the recombinant CHO cell line expressing CD80-IgG1Fc fusion protein stably was obtained. For H 22 cells modified by fusion protein, the proliferation of spleen lymphocytes increased the cytotoxic activity of CTL compared with the control group. After inoculation of H22 cells modified by fusion protein, the subcutaneous tumor volume of tumor-bearing mice was smaller than that of control group on the 12th day. Conclusion the fusion protein can be stably expressed in CHO cells, and H22 cells modified with CD80 IgG1Fc fusion protein can promote lymphocyte activation, induce specific anti-tumor immune effect, and inhibit tumor growth in vivo.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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