免疫毒素IL-18-PE38融合基因重组载体构建及其在疾病治疗中的初步应用
发布时间：2019-03-10 19:42
【摘要】：目的: 构建免疫毒素IL-18-PE38融合基因真核表达重组载体,并研究该重组质粒对IL-18受体超表达的白血病及类风湿性关节炎是否有治疗作用。方法与结果: (1) 通过限制性内切酶双酶切从质粒PRKL459K-IL18-PE38中获取IL18-PE38融合基因,将其与分泌性真核表达载体pSecTag2B连接,转化感受态菌,挑取单克隆培养并提取质粒,EcoRI单酶切后电泳鉴定显示所构建真核表达载体pSecTag2B-IL-18—PE38片段长度约为6.8kb。DNA测序结果显示IL-18和PE38序列与基因文库中报道序列相符。 (2) 脂质体转染法将构建的真核表达质粒转染入3T3细胞,荧光免疫细胞化学法荧光显微镜照片显示转染融合基因组荧光表达强,空载体对照组荧光表达微弱。转染之3T3细胞用Zeocin筛选纯系转染阳性细胞,筛选50天后,空白组细胞死亡,对照组与转染组存活细胞成克隆性生长。采用斑点ELLSA和Western-blot法鉴定细胞培养上清,证实细胞培养上清中有融合蛋白表达。将上清作用于白血病L615细胞株,MTT法测定细胞生长情况,结果显示转染重组基因组细胞死亡率高于转染空载体对照组。流式细胞术检测结果示转染重组基因组可见明显凋亡峰。AO/EB荧光双染色荧光显微镜照片显示转染组较对照组呈现明显凋亡细胞染色特征。
[Abstract]:Aim: to construct the eukaryotic expression recombinant vector of immunotoxin IL-18-PE38 fusion gene and to study the therapeutic effect of the recombinant plasmid on leukemia and rheumatoid arthritis with IL-18 receptor overexpression. Methods and results: (1) IL18-PE38 fusion gene was obtained from plasmid PRKL459K-IL18-PE38 by restriction endonuclease digestion, then ligated with secretory eukaryotic expression vector pSecTag2B and transformed into competent bacteria. The pSecTag2B-IL-18-PE38 fragment length of the constructed eukaryotic expression vector was about 6.8kb.DNA sequencing results showed that the IL-18 and PE38 sequences were consistent with the reported sequences in the gene library. The results of EcoRI single enzyme digestion and electrophoresis showed that the constructed eukaryotic expression vector pSecTag2B-IL-18-PE38 fragment length was about the same as that reported in the gene library. (2) Eukaryotic expression plasmid was transfected into 3T3 cells by lipofectamine transfection. Fluorescence immunocytochemical fluorescence microscopy showed that the fluorescent expression of fusion genome was strong, while that of empty vector control group was weak. The transfected 3T3 cells were screened by Zeocin. After 50 days of screening, the cells in the blank group died, and the surviving cells in the control group and the transfected group grew into clones. Dot ELLSA and Western-blot methods were used to identify the expression of fusion protein in the supernatant of cell culture. The cell growth of L615 cells was measured by MTT assay. The results showed that the death rate of transfected recombinant genomic cells was higher than that of blank vector control group. The results of flow cytometry showed that apoptosis peaks could be seen in the transfected recombinant genome, and AO / EB fluorescence double staining fluorescence microscope photographs showed that the transfected group showed obvious apoptotic cells staining characteristics compared with the control group.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q789;R346
本文编号：2437956
[Abstract]:Aim: to construct the eukaryotic expression recombinant vector of immunotoxin IL-18-PE38 fusion gene and to study the therapeutic effect of the recombinant plasmid on leukemia and rheumatoid arthritis with IL-18 receptor overexpression. Methods and results: (1) IL18-PE38 fusion gene was obtained from plasmid PRKL459K-IL18-PE38 by restriction endonuclease digestion, then ligated with secretory eukaryotic expression vector pSecTag2B and transformed into competent bacteria. The pSecTag2B-IL-18-PE38 fragment length of the constructed eukaryotic expression vector was about 6.8kb.DNA sequencing results showed that the IL-18 and PE38 sequences were consistent with the reported sequences in the gene library. The results of EcoRI single enzyme digestion and electrophoresis showed that the constructed eukaryotic expression vector pSecTag2B-IL-18-PE38 fragment length was about the same as that reported in the gene library. (2) Eukaryotic expression plasmid was transfected into 3T3 cells by lipofectamine transfection. Fluorescence immunocytochemical fluorescence microscopy showed that the fluorescent expression of fusion genome was strong, while that of empty vector control group was weak. The transfected 3T3 cells were screened by Zeocin. After 50 days of screening, the cells in the blank group died, and the surviving cells in the control group and the transfected group grew into clones. Dot ELLSA and Western-blot methods were used to identify the expression of fusion protein in the supernatant of cell culture. The cell growth of L615 cells was measured by MTT assay. The results showed that the death rate of transfected recombinant genomic cells was higher than that of blank vector control group. The results of flow cytometry showed that apoptosis peaks could be seen in the transfected recombinant genome, and AO / EB fluorescence double staining fluorescence microscope photographs showed that the transfected group showed obvious apoptotic cells staining characteristics compared with the control group.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q789;R346
【共引文献】
相关期刊论文 前1条
1 胡洪慧,王凤山,凌沛学;白细胞介素-4的研究进展[J];中国药学杂志;2005年10期
，本文编号：2437956
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2437956.html
最近更新
教材专著
