【作者】 索效军；

【导师】 韩俊文； 张希春；

【作者基本信息】 山西农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本研究选择草鱼作为研究对象,①对草鱼和鲤鱼GH 氨基酸序列进行比较,综合考虑选定序列有最小的同源性、最大的亲水性、最大的抗原决定簇等参数,设计出能特异性识别草鱼GH 的12 肽序列,其中11 个氨基酸来源于草鱼GH 序列,在羧基端外加1 个半胱氨酸以利于与载体连接;②预测出的抗原肽段利用Merifield固相法合成原理,在ABI430型自动多肽合成仪上合成,利用质谱仪来估测合成多肽的纯度和分子结构,多肽纯度达96%以上;③合成的多肽用MBS 法与大蛋白KLH 偶联;④将此复合肽作为抗原免疫新西兰大白兔,建立草鱼GH 多克隆抗体体系,制备出特异性强、滴度高的GH 多克隆抗体来进行免疫检测,其结果如下: Western blot 免疫分析结果显示,草鱼GH 与兔抗草鱼GH 血清能发生结合反应,在PVDF 膜上呈现一条分子量为22KD 的显色带。免疫交叉反应表明,草鱼GH 抗血清只与草鱼GH 特异结合,而与鲤鱼GH 没有交叉反应。本试验创立了抗原肽检测草鱼GH 基因的研究方法;通过抗原—抗体之间的特异性结合关系,证实所制备的抗血清能与有活性的草鱼GH发生特异性反应。应用ELISA和Western blot 技术,进行草鱼GH 基因的免疫定量研究,为建立草鱼GH转录产物和表达蛋白质的检测方法奠定了基础;为在翻译水平上检测基因的表达部位和表达量提供了一种新的手段。因此也可为建立快捷、准确、灵敏的转基因鱼检测方法提供科学依据。更多还原

【Abstract】 The study choose grass carp as the experimental object. ①Based on the deduced amino-acid sequence, gcGH were compared with known ccGH in this experiment. eleven peptides representing sequences from gcGH and an extra cystine added to its N-terminal part were chosen to synthesis based on gcGH and ccGH having minimal sequence identity, maximal hydropathy of gcGH, and maximal probability of corresponding to strongly immunogenic region of gcGH, design specific antibodies useful for detection of grass carp growth hormone;②Twelve peptides were corresponding to solid-phase methodology on an ABI 430A peptide synthesizer employing user-devised cycles the crude peptides were purified to >96% by reversed-phase HPLC with a gradient of acetonitrile in struture and purity were confirmed in all cases by electrospray ionization-mass spectrometry; ③SC-12 is a kind of hapten that must conjugate to carrier protein KLH with MBS methods;④gcGH polycolonal antibody system is established; highly titer and specifc antiserum is prepared;the results as follows: Western blot and cross reaction of gcGH and ccGH with gcGH antiserum of 1:10000 dilution confirmed that gcGH protein could specifically react with antibody against gcGH.A method that determines the immune quantification study of GH expressed has been successfully established with the antigen peptide in this study. And then by this method through the specific combination between antigen and antibody, it has been further confirmed that the anti-serum has the specific reaction to the active grass carp growth hormone. With the ELISA and Western blot technique, we carried out the immune quantification study of gcGH that will be the basis to develop a method to determine the GH and expressed protein. The result of this study can provide the scientific data to develop a rapid, accurate and sensitive detection method of grass carp, and is important to the study of molecular biology of fish growth hormone and GH gene transfer breeding.更多还原