發(fā)布時(shí)間：2018-03-11 21:29

  本文選題：牛乳　切入點(diǎn)：羊乳　出處：《陜西科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：牛乳和山羊乳作為最常見的兩種乳源,是人類重要的蛋白質(zhì)營(yíng)養(yǎng)來源。牛羊乳蛋白質(zhì)的差異往往導(dǎo)致加工特性的不同,也是區(qū)別檢測(cè)牛羊乳的主要目標(biāo)物質(zhì)。本課題以牛羊乳的蛋白質(zhì)為主要研究對(duì)象,采用單向和雙向電泳的技術(shù)分析比較了牛羊乳蛋白質(zhì)的異同,應(yīng)用蛋白組學(xué)研究技術(shù)對(duì)牛乳和山羊乳中的蛋白質(zhì)進(jìn)行分析鑒定和相應(yīng)的生物信息學(xué)分析探討,以牛乳β-乳球蛋白(β-LG)為檢測(cè)對(duì)象研究膠體金免疫層析快速檢測(cè)試紙條。本文研究?jī)?nèi)容及結(jié)果如下:牛羊乳聚丙烯酰胺凝膠電泳研究。以脫脂牛羊乳為樣品,分別進(jìn)行非變性(Native)、變性還原(SDS~R)、變性非還原(SDS~(NR))條件單向電泳以及Native:SDS~(NR)-PAGE和SDS~(NR):SDS~R-PAGE雙向電泳,比較牛羊乳圖譜異同。在Native-PAGE圖譜中,牛羊乳蛋白差異主要表現(xiàn)在β-LG條帶上,牛乳的β-LGA和β-LGB條帶可以作為區(qū)別牛羊乳的特征條帶。SDS-PAGE圖譜中,αs1-CN和αs2-CN由于含量差異而表現(xiàn)出的電泳條帶差別是區(qū)別牛羊乳的主要特征。單向電泳圖譜結(jié)合Native:SDS~(NR)-PAGE和SDS~(NR):SDS~R-PAGE雙向電泳圖譜顯示,加熱會(huì)使牛羊乳中熱敏性蛋白發(fā)生變性聚合,熱處理越劇烈,蛋白變性聚合程度越高。其中β-LG易與κ-CN以及αs2-CN以二硫鍵形式聚合形成聚合體分布于酪蛋白膠束表面以及乳清相中。而牛羊乳由于蛋白含量差異,聚合體的分布比例略有不同。加壓條件也會(huì)促使β-LG與酪蛋白的結(jié)合,羊乳由于清蛋白含量較高,此種聚合現(xiàn)象更加明顯。牛羊乳蛋白組學(xué)研究。以脫脂牛羊乳和牛羊乳清為樣品,進(jìn)行IEF:SDS-PADE雙向電泳,分別得到總蛋白和清蛋白的雙向電泳指紋圖譜。結(jié)果顯示,牛羊乳在蛋白種類上的差異并不明顯,但同種類蛋白在2-DE圖譜中的電泳行為卻不相同。牛羊乳2-DE圖譜區(qū)別較大的蛋白主要有αs1-CN、αs2-CN、κ-CN以及β-LG,其主要區(qū)別體現(xiàn)在含量上的差異、蛋白自身變異體存在以及蛋白質(zhì)磷酸化修飾導(dǎo)致的等電點(diǎn)差異。通過選定2-DE凝膠中蛋白點(diǎn)進(jìn)行酶解質(zhì)譜分析及蛋白組查庫匹配分析鑒定,確定了牛羊乳主要酪蛋白及清蛋白在2-DE圖譜中的位置及相應(yīng)蛋白質(zhì)的分子結(jié)構(gòu)信息數(shù)據(jù)。乳清蛋白中鑒定出大量的牛乳和羊乳的低豐度蛋白,比如牛乳絲氨酸蛋白酶、維生素D結(jié)合蛋白、α-1-抗糖蛋白以及羊乳α-2-HS-糖蛋白等。對(duì)牛羊乳的五種大量蛋白進(jìn)行的二級(jí)結(jié)構(gòu)預(yù)測(cè)和表位分析結(jié)果表明,牛羊乳同種蛋白抗原表位均存在差異,實(shí)現(xiàn)特定表位片段識(shí)別是牛羊乳蛋白的區(qū)別檢測(cè)基礎(chǔ)。免疫檢測(cè)技術(shù)研究。選取牛乳α-CN、α-LA、β-CN和β-LG作為抗原制備的抗體α-CN-IgG、α-LA-IgG、β-CN-IgG和β-LG-IgG的效價(jià)分別為1:204800、1:204800、1:3200和1:25600。競(jìng)爭(zhēng)ELISA法測(cè)定四種抗體的競(jìng)爭(zhēng)抑制曲線,四種抗體的半抑制濃度分別為9.2 mg/mL、1.4 mg/mL、5281.8mg/mL和48.4 mg/mL。交叉反應(yīng)測(cè)試結(jié)果顯示β-LG-IgG抗體有較高的特異性。選擇β-LG-IgG研制檢測(cè)牛乳β-LG的膠體金免疫層析試紙條,其膠體金最佳標(biāo)記pH為8.0,最佳蛋白標(biāo)記量為25μg/mL。試紙條的NC膜選擇Sartorius CN 140,金標(biāo)墊和樣品墊的封閉劑選擇PEG-20000。C線二抗的包被濃度為1.0 mg/mL,T線多抗的包被濃度為1.0 mg/mL,金標(biāo)墊上金標(biāo)抗體的涂布濃度為20%。以此條件制備的試紙條可以明確區(qū)分陰性和陽性反應(yīng),其對(duì)β-LG的檢測(cè)靈敏度可達(dá)5μg/mL。通過對(duì)牛羊乳蛋白質(zhì)的電泳分析及蛋白質(zhì)組學(xué)研究,鑒定了牛羊乳中酪蛋白、清蛋白和低豐度蛋白,分析比較了主要牛羊乳蛋白質(zhì)及抗原表位的異同�；讦�-LG的膠體金免疫層析試紙條的研究為牛羊乳快速區(qū)別檢測(cè)奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：陜西科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ937

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本文編號(hào)：1599972