贾第虫病毒转染载体的构建及绿色荧光蛋白在贾第虫体内的表达

发布时间：2018-01-14 11:13

本文关键词：贾第虫病毒转染载体的构建及绿色荧光蛋白在贾第虫体内的表达　出处：《吉林大学》2005年博士论文　论文类型：学位论文

【摘要】：本研究以中国人源贾第虫病毒(GLV)基因组为基础,构建GLV与GFP的嵌合体,优化GLV表达外源基因的顺式作用元件;克隆贾第虫GDH启动子并对其转录活性进行了鉴定;构建NEO抗性盒pGDH5/NEO/GDH3并转染贾第虫,通过G418筛选获得抗性虫株;以G418为筛选标记,构建贾第虫DNA稳定转染载体,建立DNA稳定转染方法;首次将GLV高效表达外源基因的顺式作用元件置于GDH启动子下游,构建GLV转录盒,将GLV转录盒与NEO抗性盒串联获得GLV载体pGLV,线性化pGLV后电穿孔转染贾第虫北京株,通过G418筛选建立了抗性虫株,经核酸检测发现线性质粒pGLV已整合入贾第虫基因组,而GLV和GFP的嵌合体则以双链RNA的形式存在;遗传稳定性分析表明该抗性虫株具有较好的遗传稳定性;并且证实GFP在贾第虫体内得到了表达,并且主要以分泌蛋白的形式表达,表达GFP能与兔抗GFP血清发生反应;经(NH_4)_2SO_4沉淀、凝胶过滤层析柱纯化后其纯度达85.3%,蛋白质浓度测定表明在培养上清中目的蛋白含量为0.56g/L。该表达系统具有表达量高、表达产物易于纯化的特点,从而为外源基因在真核细胞中的表达提供了新的方法。
[Abstract]:In this study, Chinese Giardia virus (GLV) genome based chimeras GLV and GFP, cis acting element optimization of GLV expression of exogenous gene; cloning of Giardia GDH promoter and its transcriptional activity were identified; construction of NEO resistance box pGDH5/NEO/GDH3 transfection and Giardia, selected by G418 resistant strains; using G418 as selection marker, construction of Giardia DNA stable transfection vector, establishment of DNA stable transfection method; first GLV exogenous gene expression of the cis elements in the GDH promoter to construct GLV transcription box, GLV box and NEO box series resistance transcription vector GLV was linearized pGLV pGLV after electroporation of Giardia isolates from Beijing, selected by G418 established resistant strains, the nucleic acid detection of linear plasmid pGLV was integrated into the Giardia genome, and chimeras of GLV and GFP in double stranded RNA form;. The analysis shows that the resistant strain had good genetic stability and transmission stability; confirmed that GFP was expressed in Giardia in vivo, and expressed mainly in the form of secreted protein, the expression of GFP and Rabbit anti GFP serum reacted with _2SO_4; (NH_4) precipitation, gel filtration chromatography the purity of the purified protein reached 85.3%. The concentration measurements show that the 0.56g/L. in the protein content in the culture supernatant of expression system with high expression amount, expression characteristics of easy purification of products, and a new method for exogenous gene expression in eukaryotic cells.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346;S852.7

【引证文献】