不同种属动物红细胞膜大豆凝集素特异性结合蛋白的分离与鉴定

发布时间：2018-03-08 18:54

本文选题：大豆凝集素　切入点：红细胞膜　出处：《吉林农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：大豆凝集素(Soybean agglutinin,SBA)是大豆中含量较高的一类具有多种生物活性的蛋白质,主要通过与细胞直接或间接的接触而发挥自身特有的生物学功能,其中一个最基本的生物学功能就是能够凝集一些动物的红细胞。已有研究表明,不同种属动物的红细胞对SBA的敏感程度不同,但具体机制尚不明确。本试验选择种属特征明显的奶牛、猪、兔的红细胞作为试验材料,研究不同动物的红细胞膜上与SBA特异结合蛋白种类的异同。试验1:采用低渗溶血法提取牛的红细胞膜,分别用NP-40裂解液、RIPA裂解液(中)和RIPA裂解液(弱)作用于牛红细胞膜,结果显示:NP-40裂解液裂解红细胞膜蛋白的效果优于RIPA裂解液(中)和RIPA裂解液(弱)。比较奶牛、猪和兔的红细胞膜蛋白SDS-PAGE电泳图可发现,三种动物红细胞的膜蛋白组成具有相似性,大部分蛋白分布在25 kDa-250 kDa之间,且高分子量的蛋白占的比例明显高于低分子量的蛋白。同时它们间也存在一定的差异性:奶牛红细胞膜蛋白最为丰富,兔红细胞膜蛋白在180 kDa和45 kDa的蛋白丰富度不如奶牛和猪,而猪红细胞膜在35 kDa处的蛋白丰富度又不如牛和兔。试验2:通过免疫共沉淀(Co-IP)方法得到红细胞膜SBA特异结合蛋白,再用液相色谱-质谱/质谱(LC-MS/MS)联用的方法对这些蛋白进行鉴定,通过MASCOT(版本号:2.3.0)软件搜库,奶牛评分大于24分、猪评分大于23分、兔评分大于22分的蛋白均为阳性结果。按照蛋白功能可将质谱得到的不同种属动物膜蛋白分为三类:第一类为细胞膜骨架蛋白,第二类为催化酶类蛋白,第三类为功能调节蛋白。其中三种动物的骨架蛋白种类均高于催化酶类和功能调节类蛋白,且评分普遍较高;在不同动物中也检测到了相同的蛋白,但大部分蛋白存在种属差异。研究结果为揭示不同种属动物红细胞对SBA敏感程度差异的机制提供了重要的试验依据。
[Abstract]:Soybean agglutininine (SBA) is a kind of protein with high content in soybean, which has a variety of biological activities. It mainly exerts its unique biological function through direct or indirect contact with cells. One of the most basic biological functions is the ability to agglutinate the erythrocytes of some animals. However, the specific mechanism is still unclear. The red blood cells of cows, pigs and rabbits with obvious species characteristics were selected as experimental materials. To study the similarities and differences between erythrocyte membrane and SBA specific binding proteins in different animals. Experiment 1: bovine erythrocyte membrane was extracted by low osmotic hemolysis method, and the bovine erythrocyte membrane was treated with NP-40 lysate (middle) and RIPA lysate (weak), respectively. The results showed that the RBC membrane protein cleavage effect of WNP-40 was better than that of RIPA lysate (middle) and RIPA cleavage solution (weakly). The results of SDS-PAGE electrophoresis of erythrocyte membrane protein in dairy cattle, pig and rabbit could be found. The membrane protein composition of erythrocytes of the three species was similar, and most of the proteins were distributed between 25 kDa-250 kDa. And the proportion of high molecular weight protein is obviously higher than that of low molecular weight protein, and there are some differences between them: cow red blood cell membrane protein is the most abundant, The protein richness of rabbit erythrocyte membrane protein at 180 kDa and 45 kDa was not as good as that of cow and pig, and the protein richness of porcine erythrocyte membrane at 35 kDa was lower than that of cattle and rabbit. Experiment 2: erythrocyte membrane SBA specific binding protein was obtained by co-immunoprecipitation method. These proteins were identified by liquid chromatography-mass spectrometry / mass spectrometry combined with LC-MS / MS.Through the software MASCOT (version No. 2.3.0), the cow score was more than 24 points, and the pig score was more than 23 points. According to the protein function, the different species of animal membrane proteins obtained by mass spectrometry can be divided into three categories: the first is cytoskeletal protein, the second is catalytic enzyme protein. The three kinds of skeleton proteins were higher than catalytic enzymes and functional regulatory proteins, and the same proteins were detected in different animals. The results provide important experimental basis for revealing the mechanism of erythrocyte sensitivity to SBA in different species of animals.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.45

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