QQ客服 官方微博 反馈留言

题名	小鼠Bicc1 基因的克隆、Bicc1 多克隆抗体制备及蛋白定位与表达的研究
作者	周亮
学位类别	硕士
答辩日期	2009-06
授予单位	中国科学院研究生院
授予地点	北京
导师	吴冠青 ; 杨君兴
关键词	Bicc1 克隆 原核表达 多克隆抗体 表达水平 RNAi
其他题名	CLONING OF THE MOUSE BICAUDUAL-C-1 GENE 、PREPARATION OF POLYCLONAL ANTIBODY 、 THE LOCATION AND EXPRESSION OF BICC1
中文摘要	双尾-C基因 (Bicaudal-C)首先在果蝇（Drosophila melanogaster）中发现，其功能丧失导致果蝇胚胎滤泡细胞的错误迁移、头部的缺失和双尾结构的形成。后来发现多个物种都含有Bicaudal-C的同源基因，其中小鼠中的同源基因Bicc1的缺失导致小鼠产生肾脏等脏器的病变，其症状与人类多囊肾疾病高度相似，但其具体机制还不清楚。 本研究以小鼠肾脏组织总RNA为模板体外反转录为cDNA，通过分段巢式PCR 及酶切连接的方法获得了全长约3Kb的小鼠Bicc1 cDNA序列。 根据生物信息学分析全长的Bicc1蛋白，选择两个免疫原性较好的区段作为抗原位点构建相应的原核表达载体；IPTG诱导表达并纯化融合蛋白，制备两种兔抗Bicc1蛋白多克隆抗体，并通过Western blot证实这两种抗体具有高度特异性。 用细胞免疫荧光方法及免疫组织化学方法对该蛋白的定位做了一些初步研究。发现Bicc1蛋白定位于体外培养的小鼠肾细胞的细胞质内，并在胚胎发育于期表达仅在心脏，后来逐步地在各个组织器官内出现，并在出生后的小鼠体内表达稳定。Bicc1 mDNA也表达于多个器官内，并且在肾脏中有明显较高的表达量。 找到了的两个针对Bicc1基因的RNAi的序列，通过荧光强度变化和Western blot均证明这两个序列能明显降低Bicc1蛋白在体外培养细胞中的表达水平，为下一步建立稳定的细胞株奠定了良好的基础。
英文摘要	The Bicaudal-C (Bic-C) gene was first discovered in Drosophila melanogaster, it’s normal function is required for correct targeting of the migrating anterior follicle cells and for anterior-posterior patterning of the Drosophila oocyte. Mouse Bicc1 gene is a homologe of Drosophila Bicaudal-C gene, whose mutation causes a phenotype sembles with human polycystic kidney disease, but the details of Bicc1 involved in cystogenesis are still elusive. Here we cloned the full-length cDNA sequence of Bicc1 gene by nest PCR and digest- ligate methods. Based on the bioinformatics analysis results of mouse Bicc1, the DNA fragment encoding N-terminal 139 amino acids(61E-199A) and C-terminal 148 amino acids(711Q-858D) of Bicc-1 was obtained by PCR and was cloned into a prokaryotic GST-fusion protein expression vector. Fusion protein were expressed after IPTG induction, and then purified from the total proteins of E. coli Rosetta cells transformed by the recombinant plasmids pGex-HisC3-GST- Bicc1-N/C. We prepared two polyclone antibodies against mouse Bicc1 protein with high specificity by immunizing rabbit with these two purified fusion proteins. The specificity of the affinity-purified antibodies was iedntified by Western blot. We stained cultured cells and mouse kidney paraffin sections with this antibody and found that Bicc1 is mainly localized at cytoplasm in cultured mouse kidney cells. And Bicc1fristly just located in heart and neural tube in early mouse embryo, then found in many different tissues and keep stable expression level. Bicc1 mDNA expess in different organs and mainly in kidney. Get two RNAi sequences that specifically knock-down Bicc1expression level in cultured cells by fluorescence and Western-blot, provide the tools for the research in low-expression stable cell lines.
语种	中文
公开日期	2010-10-22
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6293]
专题	昆明动物研究所_系统进化与生物地理学
推荐引用方式 GB/T 7714	周亮. 小鼠Bicc1 基因的克隆、Bicc1 多克隆抗体制备及蛋白定位与表达的研究[D]. 北京. 中国科学院研究生院. 2009.

个性服务

查看访问统计

相关权益政策

暂无数据

收藏/分享

除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。