【作者】 李晶；

【导师】 安铁洙；

【作者基本信息】 东北林业大学 ， 发育生物学， 2010， 硕士

【摘要】 为了建立通过哺乳动物被毛获取蜘蛛丝的方法,本研究以小鼠为动物模型,利用研究室前期工作中获得的蜘蛛拖丝蛋白二聚体、小鼠皮肤特异性启动子UHS和构建的UHS-2S-3.1重组质粒,构建在机体中广泛表达的CMV-2S-pIRES2-EGFP质粒和在皮肤上特异表达且可通过报告基因GFP便于检测的UHS-2S-pIRES2-EGFP重组质粒,分别在转染小鼠颗粒细胞和小鼠胎儿皮肤成纤维细胞基础上,通过G418筛选并经PCR法鉴定,检测其在基因组中整合情况。其结果：(1)蜘蛛拖丝蛋白基因真核表达载体的构建：将二聚化拖丝蛋白基因与载体pIRES2-EGFP相连接,获得表达载体CMV-2S-pIRES2-EGFP;通过特异性引物将角蛋白启动子UHS扩增,连接到切除自身CMV病毒启动子的CMV-2S-pIRES2-EGFP载体上,获得拖丝蛋白基因真核表达载体UHS-2S-pIRES2-EGFP。(2)颗粒细胞的转染与筛选：利用幼鼠颗粒细胞,通过体外培养,可获得最佳的原代贴壁时间、传代速率、细胞形态和冷冻复苏后活性等指标的细胞株；用线性化的UHS-2S-3.1载体转染到颗粒细胞后,经G418筛选并通过PCR鉴定证实,细胞基因组中整合含有UHS-2S-3.1。(3)成纤维细胞的转染与筛选：采用常规方法建立小鼠皮肤成纤维细胞系；用线性化的CMV-2S-pIRES2-EGFP以及UHS-2S-pIRES2-EGFP载体转染成纤维细胞并进行G418抗性筛选扩增的阳性细胞,经对其基因组DNA的PCR鉴定,确认基因组整合均带有目的基因。上述研究结果,为利用UHS-2S-pIRES2-EGFP重组质粒,采用原核显微注射法或者通过体细胞核移植法获得在被毛特异表达蜘蛛拖丝蛋白的转基因鼠,最终建立通过绵羊被毛获取蜘蛛拖丝蛋白的新方法奠定基础。更多还原

【Abstract】 In order to establish a new method to obtain the spider dragline silk from animal hair, mouse was chose as a animal model. By using spider dragline silk protein which was got from the preliminary work of the laboratory, mouse skin-specific promoter and recombinant plasmid named UHS-2S-3.1,CMV-2S-pIRES2-EGFP plasmid which is widely expressed in the body and the UHS-2S-pIRES2-EGFP plasmid which is not only expressed specifically in the skin but also can be easily detected by GFP reporter gene were constructed. Then mouse granular cells and mouse fetal skin fibroblasts were transfected by the recombinant plasmids respectively. G418 was used to filtrate the transfected cells and the anti-G418 cells were identified by PCR method to showed whether the vector constructed was integrated into cells genome. Results showed as follows:1.Construction of eukaryotic expression vector with silk dragline protein gene: polymerized spider dragline silk protein gene was linked to pIRES2-EGFP plasmid to get the expression vector CMV-2S-pIRES2-EGFP. Mouse skin-specific promoter gene was linked to the vector of CMV-2S-pIRES2-EGFP that wiped off CMV promoter after amplification in order to obtain the eukaryotic expression vectors with dragline silk protein UHS-2S-pIRES2-EGFP.2. Transfection and Filtration of mouse granular cells:granular cells from the young mouses were cultured in vitro to get the optimal cell line; After transfected linearization vector UHS-2S-3.1, the granular cells were filtrated by G418 and anti-G418 cells were identified by PCR method, the result showed that the vector constructed was integrated into granular cells genome.3. Transfection and Filtration of mouse fibroblasts:mouse fibroblast cell line were established by conventional methods; mouse skin fibroblasts were transfected with linearization vector CMV-2S-pIRES2-EGFP and UHS-2S-pIRES2-EGFP,then G418 was used to filtrate them. anti-G418 cells were identified by PCR and PCR identification of the positive cells indicated that objective gene integrated into genomic.These results of using original nuclear microinjection method or somatic cell nuclear transfer technology to get transgenic mouse which hair expressed spider dragline silk protein gene are conducive to establish a new method to get spider dragline silk protein gene from sheep hair. in the further research.更多还原