牛羊乳蛋白质的双向电泳分析及免疫检测技术研究

发布时间：2018-03-11 21:29

本文选题：牛乳　切入点：羊乳　出处：《陕西科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：牛乳和山羊乳作为最常见的两种乳源,是人类重要的蛋白质营养来源。牛羊乳蛋白质的差异往往导致加工特性的不同,也是区别检测牛羊乳的主要目标物质。本课题以牛羊乳的蛋白质为主要研究对象,采用单向和双向电泳的技术分析比较了牛羊乳蛋白质的异同,应用蛋白组学研究技术对牛乳和山羊乳中的蛋白质进行分析鉴定和相应的生物信息学分析探讨,以牛乳β-乳球蛋白(β-LG)为检测对象研究胶体金免疫层析快速检测试纸条。本文研究内容及结果如下:牛羊乳聚丙烯酰胺凝胶电泳研究。以脱脂牛羊乳为样品,分别进行非变性(Native)、变性还原(SDS~R)、变性非还原(SDS~(NR))条件单向电泳以及Native:SDS~(NR)-PAGE和SDS~(NR):SDS~R-PAGE双向电泳,比较牛羊乳图谱异同。在Native-PAGE图谱中,牛羊乳蛋白差异主要表现在β-LG条带上,牛乳的β-LGA和β-LGB条带可以作为区别牛羊乳的特征条带。SDS-PAGE图谱中,αs1-CN和αs2-CN由于含量差异而表现出的电泳条带差别是区别牛羊乳的主要特征。单向电泳图谱结合Native:SDS~(NR)-PAGE和SDS~(NR):SDS~R-PAGE双向电泳图谱显示,加热会使牛羊乳中热敏性蛋白发生变性聚合,热处理越剧烈,蛋白变性聚合程度越高。其中β-LG易与κ-CN以及αs2-CN以二硫键形式聚合形成聚合体分布于酪蛋白胶束表面以及乳清相中。而牛羊乳由于蛋白含量差异,聚合体的分布比例略有不同。加压条件也会促使β-LG与酪蛋白的结合,羊乳由于清蛋白含量较高,此种聚合现象更加明显。牛羊乳蛋白组学研究。以脱脂牛羊乳和牛羊乳清为样品,进行IEF:SDS-PADE双向电泳,分别得到总蛋白和清蛋白的双向电泳指纹图谱。结果显示,牛羊乳在蛋白种类上的差异并不明显,但同种类蛋白在2-DE图谱中的电泳行为却不相同。牛羊乳2-DE图谱区别较大的蛋白主要有αs1-CN、αs2-CN、κ-CN以及β-LG,其主要区别体现在含量上的差异、蛋白自身变异体存在以及蛋白质磷酸化修饰导致的等电点差异。通过选定2-DE凝胶中蛋白点进行酶解质谱分析及蛋白组查库匹配分析鉴定,确定了牛羊乳主要酪蛋白及清蛋白在2-DE图谱中的位置及相应蛋白质的分子结构信息数据。乳清蛋白中鉴定出大量的牛乳和羊乳的低丰度蛋白,比如牛乳丝氨酸蛋白酶、维生素D结合蛋白、α-1-抗糖蛋白以及羊乳α-2-HS-糖蛋白等。对牛羊乳的五种大量蛋白进行的二级结构预测和表位分析结果表明,牛羊乳同种蛋白抗原表位均存在差异,实现特定表位片段识别是牛羊乳蛋白的区别检测基础。免疫检测技术研究。选取牛乳α-CN、α-LA、β-CN和β-LG作为抗原制备的抗体α-CN-IgG、α-LA-IgG、β-CN-IgG和β-LG-IgG的效价分别为1:204800、1:204800、1:3200和1:25600。竞争ELISA法测定四种抗体的竞争抑制曲线,四种抗体的半抑制浓度分别为9.2 mg/mL、1.4 mg/mL、5281.8mg/mL和48.4 mg/mL。交叉反应测试结果显示β-LG-IgG抗体有较高的特异性。选择β-LG-IgG研制检测牛乳β-LG的胶体金免疫层析试纸条,其胶体金最佳标记pH为8.0,最佳蛋白标记量为25μg/mL。试纸条的NC膜选择Sartorius CN 140,金标垫和样品垫的封闭剂选择PEG-20000。C线二抗的包被浓度为1.0 mg/mL,T线多抗的包被浓度为1.0 mg/mL,金标垫上金标抗体的涂布浓度为20%。以此条件制备的试纸条可以明确区分阴性和阳性反应,其对β-LG的检测灵敏度可达5μg/mL。通过对牛羊乳蛋白质的电泳分析及蛋白质组学研究,鉴定了牛羊乳中酪蛋白、清蛋白和低丰度蛋白,分析比较了主要牛羊乳蛋白质及抗原表位的异同。基于β-LG的胶体金免疫层析试纸条的研究为牛羊乳快速区别检测奠定了基础。
[Abstract]:Milk and goat milk as the two most common in Ruyuan, is an important source of human protein nutrition. The difference of cattle and sheep milk proteins often leads to different processing characteristics, the main difference is the target substance detection of cattle and sheep milk. The cattle and sheep milk protein as the main object of study, using the technology of unidirectional and bidirectional electrophoresis comparison analysis the similarities and differences of cattle and sheep milk protein, the application of proteomics technology was analyzed and the corresponding bioinformatics analysis of cow milk and goat milk in the milk protein, beta lactoglobulin (beta -LG) for detecting objects based on colloidal gold immunochromatographic test strip. The contents and results in this paper are as follows sheep milk: polyacrylamide gel electrophoresis. The defatted milk of cattle and sheep as samples, were non denaturing (Native), (SDS~R), degeneration degeneration reduction of non reducing (SDS~ (NR)) one-way electric Swimming and Native:SDS~ (NR) -PAGE and SDS~ (NR): SDS~R-PAGE electrophoresis, compared to cattle and sheep milk of similarities and differences. In Native-PAGE map, cattle and sheep milk protein were mainly in beta -LG bands, milk beta -LGA and beta -LGB bands can be used as a distinguishing characteristic of cattle and sheep milk with.SDS-PAGE map, a alpha s1-CN alpha s2-CN and electrophoresis due to differences in the content and the difference is mainly with the distinguishing characteristic of cattle and sheep milk. The one-way electrophoresis combined with Native:SDS~ (NR) -PAGE and SDS~ (NR): SDS~R-PAGE two-dimensional gel electrophoresis maps of heating can make the heat sensitive protein and milk denaturation polymerization, heat treatment is more intensive, more high protein denaturation degree of polymerization. The Beta Kappa -CN alpha and -LG and s2-CN to form two disulfide bonds in polymer polymerization to form distributed in the casein micelle surface and whey phase. The cattle milk protein content due to differences in the distribution proportion of the slight polymerization Different. With pressurized condition will also promote beta -LG and casein, milk because albumin content is high, this phenomenon is more obvious in cattle and sheep milk. Polymerization of proteomics research. In the skim milk and beef cattle and sheep whey samples by IEF:SDS-PADE electrophoresis, two-dimensional electrophoresis fingerprint of total protein and albumin were obtained. The results show that is not significant differences in the types of cattle and sheep milk protein, but the same kind of protein in the 2-DE Atlas of the electrophoretic behavior is not the same. Cattle and sheep milk 2-DE of the large difference between the protein mainly alpha s1-CN, alpha s2-CN, Beta Kappa -CN and -LG, the main difference reflects differences in the content of protein and protein phosphorylation of its variants modification to the isoelectric point difference. Enzyme mass spectrometry and protein group inventory matching analysis identified by selected protein 2-DE gel, determine the main cattle milk and casein The molecular structure information data of albumin in 2-DE map locations and the corresponding proteins were identified. A large number of low abundance protein of cow and goat milk whey protein, such as milk serine protease, vitamin D binding protein, alpha -1- alpha -2-HS- and Goat anti glycoprotein glycoprotein. Prediction of two level structure of five kinds of large amounts of protein for cattle and sheep milk the epitope analysis results showed that the cattle and sheep milk the same protein epitopes were different, specific epitope fragment identification is the difference between cattle and sheep milk protein based detection. Detection of immune milk. Selection of alpha -CN, alpha -LA, alpha -CN-IgG antibody, beta -CN and beta -LG as antigen preparation of alpha -LA-IgG respectively, the titer of beta -CN-IgG and beta -LG-IgG for the determination of four kinds of antibody 1:204800,1:204800,1:3200 and 1:25600. competitive ELISA competitive inhibition curve, half inhibitory concentration of four antibodies were 9.2 mg/ mL, 1.4 m G/mL, 5281.8mg/mL and mg/mL. 48.4 cross reaction test results show that the specific beta -LG-IgG antibody had higher. Colloidal gold immunochromatographic beta beta -LG -LG-IgG for the detection of milk, the optimal labeling pH of colloidal gold was 8, the best NC membrane protein markers: 25 g/mL. strip Sartorius CN 140, Kim standard sample pad pad and sealant PEG-20000.C line two antibody concentration for coating was 1 mg/mL, T line polyclonal antibody concentration for coating was 1 mg/mL, gold labeled strip mat gold labeled antibody were coated by 20%. taking the conditions of the preparation of the article and can clearly discriminate between positive reaction, electrophoresis analysis and proteomics. The cattle and sheep milk protein studies of beta -LG detection sensitivity can reach 5 mu g/mL. by cattle and sheep milk casein, albumin and identification of low abundance proteins, analysis the similarities and differences between the main cattle and sheep milk protein and epitope. The study of colloidal gold immunochromatography paper based on beta -LG lays a foundation for rapid differential detection of cow and sheep milk.

【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ937

【参考文献】