本文選題：大豆凝集素　切入點：紅細(xì)胞膜　出處：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：大豆凝集素(Soybean agglutinin,SBA)是大豆中含量較高的一類具有多種生物活性的蛋白質(zhì),主要通過與細(xì)胞直接或間接的接觸而發(fā)揮自身特有的生物學(xué)功能,其中一個最基本的生物學(xué)功能就是能夠凝集一些動物的紅細(xì)胞。已有研究表明,不同種屬動物的紅細(xì)胞對SBA的敏感程度不同,但具體機制尚不明確。本試驗選擇種屬特征明顯的奶牛、豬、兔的紅細(xì)胞作為試驗材料,研究不同動物的紅細(xì)胞膜上與SBA特異結(jié)合蛋白種類的異同。試驗1:采用低滲溶血法提取牛的紅細(xì)胞膜,分別用NP-40裂解液、RIPA裂解液(中)和RIPA裂解液(弱)作用于牛紅細(xì)胞膜,結(jié)果顯示:NP-40裂解液裂解紅細(xì)胞膜蛋白的效果優(yōu)于RIPA裂解液(中)和RIPA裂解液(弱)。比較奶牛、豬和兔的紅細(xì)胞膜蛋白SDS-PAGE電泳圖可發(fā)現(xiàn),三種動物紅細(xì)胞的膜蛋白組成具有相似性,大部分蛋白分布在25 kDa-250 kDa之間,且高分子量的蛋白占的比例明顯高于低分子量的蛋白。同時它們間也存在一定的差異性:奶牛紅細(xì)胞膜蛋白最為豐富,兔紅細(xì)胞膜蛋白在180 kDa和45 kDa的蛋白豐富度不如奶牛和豬,而豬紅細(xì)胞膜在35 kDa處的蛋白豐富度又不如牛和兔。試驗2:通過免疫共沉淀(Co-IP)方法得到紅細(xì)胞膜SBA特異結(jié)合蛋白,再用液相色譜-質(zhì)譜/質(zhì)譜(LC-MS/MS)聯(lián)用的方法對這些蛋白進行鑒定,通過MASCOT(版本號:2.3.0)軟件搜庫,奶牛評分大于24分、豬評分大于23分、兔評分大于22分的蛋白均為陽性結(jié)果。按照蛋白功能可將質(zhì)譜得到的不同種屬動物膜蛋白分為三類:第一類為細(xì)胞膜骨架蛋白,第二類為催化酶類蛋白,第三類為功能調(diào)節(jié)蛋白。其中三種動物的骨架蛋白種類均高于催化酶類和功能調(diào)節(jié)類蛋白,且評分普遍較高;在不同動物中也檢測到了相同的蛋白,但大部分蛋白存在種屬差異。研究結(jié)果為揭示不同種屬動物紅細(xì)胞對SBA敏感程度差異的機制提供了重要的試驗依據(jù)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.45

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