CD59配体肽基因真核表达体系的构建及对前列腺癌PC-3细胞活性的作用研究

发布时间：2018-03-03 13:03

  本文选题：配体　切入点：CD59　出处：《青岛大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的构建CD59分子配体肽(sp22)基因真核表达重组体系,探讨sp22基因对细胞CD59分子表达及功能的影响,为进一步研究sp22分子与CD59分子的作用机制及利用短肽封闭CD59分子表达,为肿瘤基因靶向治疗开辟新途径。方法构建含特异性sp22基因的真核重组表达载体sp-pIRES,脂质体法转染人前列腺癌PC-3细胞,G418筛选建立稳定转染细胞系,RT-PCR检测转染细胞中sp22基因mRNA的表达并筛选阳性细胞克隆,RT-PCR检测转染细胞表面CD59mRNA表达量的变化,Western Blot、ELISA竞争抑制试验检测转染细胞CD59蛋白的表达；制备抗CD59多克隆抗体,台盼蓝染色试验和LDH试验检测sp22分子对CD59分子功能的影响,MTT法测定PC-3细胞增殖抑制率。 结果PCR、双酶切及DNA测序鉴定表明成功构建了sp-PIRES真核重组表达载体；RT-PCR试验证实sp22在spPC-3细胞中成功表达；Western Blot、ELESA竞争抑制试验表明：spPC-3细胞比正常PC-3细胞CD59蛋白表达量减少,二者比较其差别有统计学意义(P0.05)；与正常PC-3细胞相比,补体对spPC-3细胞的溶解杀伤能力明显增强,二者比较其差别有统计学意义(P0.05); MTT结果测定spPC-3细胞与正常PC-3细胞相比较细胞增殖抑制率增高,差别有统计学意义(P0.05)。 结论sp22基因可在mRNA及蛋白水平抑制人前列腺癌PC-3细胞表面CD59蛋白分子的表达及功能,降低CD59分子抗补体活性,为进一步研究利用SP22阻断CD59分子在肿瘤细胞上的表达创造了有利条件,为肿瘤的免疫治疗研究奠定基础。
[Abstract]:Objective to construct a recombinant eukaryotic expression system of CD59 ligand peptide sp 22) and to investigate the effect of sp22 gene on the expression and function of CD59 molecule. In order to further study the interaction mechanism between sp22 molecule and CD59 molecule and block the expression of CD59 molecule by short peptide. Methods Eukaryotic recombinant expression vector sp-pIRESwith specific sp22 gene was constructed and transfected into human prostate cancer PC-3 cell line G418 by liposome method to establish a stable transfection cell line to detect the expression of SP-pIRESin the transfected cells by reverse transcription-polymerase chain reaction (RT-PCR). The expression of sp22 gene mRNA and the expression of CD59mRNA on transfected cells were detected by RT-PCR. The expression of CD59 protein in transfected cells was detected by Western blottELISA competitive inhibition assay. Anti CD59 polyclonal antibody was prepared, trypan blue staining and LDH assay were used to detect the effect of sp22 on the function of CD59. The inhibitory rate of PC-3 cell proliferation was determined by MTT assay. Results PCR, double enzyme digestion and DNA sequencing showed that the sp-PIRES eukaryotic expression vector was successfully constructed. RT-PCR assay confirmed that sp22 was successfully expressed in spPC-3 cells. The results of competitive inhibition test showed that the expression of CD59 protein in PC-3 cells was lower than that in normal PC-3 cells. Compared with normal PC-3 cells, the cytotoxicity of complement to spPC-3 cells was significantly increased. Compared with normal PC-3 cells, the proliferation inhibition rate of spPC-3 cells was higher than that of normal PC-3 cells, and the difference was statistically significant (P 0.05). Conclusion sp22 gene can inhibit the expression and function of CD59 protein molecules on the surface of human prostate cancer PC-3 cells at the level of mRNA and protein, and decrease the anti-complement activity of CD59 molecule. It provides a favorable condition for the further study of SP22 blocking the expression of CD59 molecules on tumor cells and lays a foundation for the study of tumor immunotherapy.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R737.25;R392

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