本文選題：CD80 + IgG1　； 參考：《福建醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:構(gòu)建人CD80-IgG1Fc融合蛋白的真核表達(dá)載體,并轉(zhuǎn)染CHO細(xì)胞,建立CHO融合蛋白穩(wěn)定表達(dá)株;探討經(jīng)融合蛋白修飾的肝癌細(xì)胞H22對淋巴細(xì)胞抗腫瘤免疫的影響,及其對肝癌細(xì)胞H22皮下移植型荷瘤小鼠腫瘤生長的影響。 方法:通過RT-PCR從人外周血單個核細(xì)胞中擴(kuò)增CD80膜外段及IgG1Fc基因,構(gòu)建真核表達(dá)載體CD80-IgG1Fc/pcDNA3.1(+),將其轉(zhuǎn)染CHO細(xì)胞株并篩選。通過RT-PCR、Western blot及ELISA鑒定融合基因的表達(dá);將融合蛋白修飾小鼠肝癌細(xì)胞株H22,流式細(xì)胞術(shù)檢測細(xì)胞膜上CD80的表達(dá),以MTT比色法、乳酸脫氫酶釋放試驗檢測經(jīng)修飾的H22細(xì)胞對脾淋巴細(xì)胞增殖及其細(xì)胞毒性的影響。建立肝癌細(xì)胞H22皮下移植型荷瘤小鼠模型后,接種經(jīng)融合蛋白修飾的H22細(xì)胞,觀察其對腫瘤生長的影響。 結(jié)果:成功構(gòu)建了真核表達(dá)載體CD80-IgG1Fc/pcDNA3.1(+),并獲得穩(wěn)定表達(dá)CD80-IgG1Fc融合蛋白的重組CHO細(xì)胞株。針對融合蛋白修飾后的H22細(xì)胞,脾淋巴細(xì)胞增殖較對照組增強(qiáng),CTL的細(xì)胞毒活性增高。荷瘤小鼠接種經(jīng)融合蛋白修飾的H22細(xì)胞后,皮下腫瘤體積在第12天小于對照組。 結(jié)論:CD80 -IgG1Fc融合蛋白能在CHO細(xì)胞中穩(wěn)定表達(dá);用CD80 -IgG1Fc融合蛋白修飾的H22細(xì)胞能促進(jìn)淋巴細(xì)胞活化,誘導(dǎo)特異性抗腫瘤免疫效應(yīng),并對體內(nèi)腫瘤生長有抑制作用。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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本文編號：1947641