【作者】 蒋朋飞；

【导师】 张德礼；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2010， 硕士

【摘要】 细胞凋亡(apoptosis)也称为“程序性细胞死亡”或“细胞自杀”,是由基因介导的一系列变化,是存在于所有哺乳动物细胞中的保守途径。细胞凋亡是调节生物体正常发育和生命活动的一种不可缺少的机制,该调节一旦失败,可能导致机体疾病、畸形甚至死亡。对免疫系统而言,细胞凋亡不仅是免疫系统发育过程中必不可少的一个环节,而且是免疫系统行使功能的一种方式。细胞凋亡的发生和调控涉及到多个基因家族,其中BCL2基因家族就是一个与凋亡密切相关的基因家族,该家族成员对细胞凋亡与否起关键作用。BCL-G也称作BCL2-like14,是近来在人类,小鼠,牛等物种发现的BCL2家族的成员之一,据研究证明该成员为促细胞凋亡因子。本研究根据生物进化论和基因存在论,利用电子克隆和实验克隆在猪体内克隆出该基因。然后,将该基因序列构建到原核及真核表达载体,进行原核表达,纯化原核目的蛋白制备了其多克隆抗体;进行真核表达,并检测了抗体的特异性。本研究获得以下实验结果。1.根据生物进化论和基因存在论,利用电子克隆,以人类BCL-G基因序列作为种子序列,进行同源比对,得到猪的同源基因的cDNA序列。进行基因特征分析发现,该基因的开放读码框(ORF)区包含990个核苷酸,编码329个氨基酸,起始密码子ATG上游同一相位有多个终止密码子TGA,终止密码子下游具有polyA尾巴。经外显子分析发现,该基因共有6个外显子,5个内含子。2.将氨基酸序列进行蛋白质的同源比对,发现与牛类的BCL-G蛋白最相似,同源度为75%。进一步对其进行重要蛋白基序和结构域进行预测,发现该蛋白具有BCL2家族的典型结构域BH2和BH3以及骨唾液酸蛋白Ⅱ结构域。3.根据电子克隆得到的核酸序列,进行引物设计,从猪的脾脏中提取总RNA作为模板,进行RT-PCR扩增得到目的基因,经测序验证,与电子克隆结果显示有3个核苷酸不同,相似度达到99%,但翻译成氨基酸序列后,相似度为100%,从而确定该基因在猪体内的存在。4.将目的基因构建到原核表达载体pET-32a(+),在大肠杆菌BL21(DE3)中进行原核表达,提取目的蛋白,免疫豚鼠,制备出多克隆抗体,并进行ELISA分析检测抗体效价;将目的基因构建到真核表达载体pEGFP-C1,经测序验证后,提取重组质粒转染猪脐静脉血管内皮细胞,利用Western blot分析检测抗体特异性。5.分别将BH2结构域缺失、BH3结构域缺失、BH23双结构域缺失的猪BCL-G_L基因构建到真核表达载体pEGFP-C1和pcDNA3.1(+)。为进一步研究该基因在真核细胞以及机体中的作用奠定了基础。更多还原

【Abstract】 Apotosis,named as programmed cell death or cell suicide,is an array of changes which are induced by many genes. It is a conservative pathway that exists extensively in all mammalian cells. Apoptosis is a necessary mechanism that regulates the development of organism. Once the regulation failed, sickness, abnormality or death may be induced. For the immune system, apoptosis is not only an indispensable segment in its development but also a manner in which the immune system functions. Many gene families are involved during the beginning and the regulation of apoptosis, in which BCL2 family is one of the gene families that link closely with apoptosis. BCL-G, also named as BCL2-like 14, belongs to the BCL2 family, was identified from a couple of organisms, such as human, mouse and cattle. It was proven to be a pro-apoptotic protein. The new gene BCL-G_L in swine was firstly cloned in silico and then was cloned by experiments according to Evolutionism and Gene Ontology. Then, swine BCL-G_L was cloned into prokaryotic expression vector pET-32a(+) and eukaryotic expressing vectors pEGFP-C1 and pcDNA3.1(+). The prokaryotic protein BCL-G_L was extracted for the preparation of its polyclonal antibody. A number of results were obtained, as follows:1. According to Evolutionism and Gene Ontology theory, Blast was made in swine EST database using human BCL-G sequence as a seed and the swine BCL-G sequence was obtained, and its characteristics were also analysed. The results show that the ORF of the gene contains 990 nucleotides and can be translated into 329 amino acids. There are couples of terminal codes in the upstream of ORF and there are polyA tails in the downstream of the ORF, the gene contains 6 exons and 5 introns.2. The amino acid sequence was blasted with the homologous proteins of other organisms. The results show that the similarity to the homologous protein of cattle is up to 75%, and the conservative domains BH2 and BH3 of BCL2 family were found in the protein.3. The primers was made according to the in silico sequence and the cloning sequence was obtained by RT-PCR from swine spleen. The cloning sequence was identified by sequencing and compared with the contig sequence.4. Swine BCL-G_L was cloned into prokaryotic expression vector pET-32a(+), and expressed in E.coli.BL21(DE3). The prokaryotic protein BCL-G_L was extracted to immunize the guinea pigs for preparing its polyclonal antibody. The titer of the polyclonal antibody was tested by ELISA; Swine BCL-G_L was cloned into eukaryotic expression vectors pEGFP-C1 and the reconstructed vectors were transfected into swine umbilical vein endothelial cells. Then, the specifity was tested by western blot assay.5. BH2 domain deficient, BH3 domain deficient and BH23 domains deficient BCL-G_L gene was cloned into eukaryotic expression vectors pEGFP-C1 or pcDNA3.1(+) in order to investigate the function of swine gene BCL -G_L.更多还原