本文關(guān)鍵詞：大豆和牛奶致敏原表位預(yù)測(cè)及加工對(duì)其抗原性和結(jié)構(gòu)的影響　出處：《江南大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 過(guò)敏原 表位預(yù)測(cè) 加工處理 抗原性 結(jié)構(gòu)變化

【摘要】：大豆和牛奶過(guò)敏在兒童和成年人中的發(fā)病率都很高，目前對(duì)于牛奶致敏原的表位了解的比較清楚，而對(duì)大豆β-伴大豆球蛋白α亞基的致敏機(jī)理尚不完全清楚，主要因?yàn)槭侨狈?duì)其致敏表位的認(rèn)識(shí)。因此，本論文借助DNAStar軟件，the BepiPred和SOPMA這三種不同的生物軟件構(gòu)建表位預(yù)測(cè)方法，通過(guò)預(yù)測(cè)大豆和牛奶已知表位的致敏原來(lái)驗(yàn)證預(yù)測(cè)方法的準(zhǔn)確性；并將該方法成功應(yīng)用于預(yù)測(cè)β-伴大豆球蛋白α亞基表位，并對(duì)預(yù)測(cè)得到的表位進(jìn)行合成，然后使用點(diǎn)雜交方法對(duì)合成的表位進(jìn)行驗(yàn)證，得到β-伴大豆球蛋白α亞基的表位。另外，通過(guò)對(duì)β-伴大豆球蛋白熱加工和α-酪蛋白脈沖處理，探討熱加工處理和脈沖處理分別對(duì)β-伴大豆球蛋白和α-酪蛋白結(jié)構(gòu)和抗原性的影響，為進(jìn)一步開(kāi)展低過(guò)敏性或無(wú)過(guò)敏性的大豆和牛奶制品加工提供理論和技術(shù)依據(jù)，主要研究結(jié)果及結(jié)論如下： 1. DNAStar，the BepiPred1.0Server，SOPMA網(wǎng)絡(luò)服務(wù)器三種生物信息學(xué)軟件成功預(yù)測(cè)得到β-伴大豆球蛋白α亞基的15段表位。使用人大豆過(guò)敏血清作為抗體，點(diǎn)雜交技術(shù)驗(yàn)證預(yù)測(cè)得到的表位，其中11段肽片段被認(rèn)為是主要人源IgE線性表位。肽序列HEQREEQEWPRKEEKRGEKGSEEEDE是α亞基最主要的人源IgE表位。使用三種軟件預(yù)測(cè)得到的表位準(zhǔn)確率較高，為表位的預(yù)測(cè)提供一種新方法。 2.為進(jìn)一步驗(yàn)證上述方法的準(zhǔn)確性，將該方法應(yīng)用于已知表位的過(guò)敏原的預(yù)測(cè)。使用三種生物信息學(xué)軟件DNAStar，SOPMA和the BepiPred對(duì)大豆Gly m Bd30K以及α-酪蛋白的線性表位進(jìn)行預(yù)測(cè)，同時(shí)結(jié)合Swiss-model對(duì)這兩種蛋白的三級(jí)結(jié)構(gòu)進(jìn)行了初步分析，得到Gly m Bd30K的4個(gè)表位區(qū)域以及α-酪蛋白的5個(gè)表位區(qū)域。將預(yù)測(cè)得到的表位與已知的表位進(jìn)行了比較，結(jié)果大豆Gly m Bd30K預(yù)測(cè)表位的正確率為75%，α-酪蛋白的正確率為60%，為致敏原表位預(yù)測(cè)奠定基礎(chǔ)。 3.將純化得到具有生物活性的β-伴大豆球蛋白α亞基免疫新西蘭大白兔得到β-伴大豆球蛋白α亞基的多克隆抗體。將多克隆抗體應(yīng)用于ELISA，檢測(cè)熱處理前后β-伴大豆球蛋白α亞基抗原性的變化；使用圓二色譜，熒光光譜以及紫外光譜檢測(cè)其結(jié)構(gòu)變化。結(jié)果發(fā)現(xiàn)低溫（50℃-70℃）短時(shí)間（35min以內(nèi)）加熱處理β-伴大豆球蛋白α亞基的抗原性是升高的，這可能是由于低溫使蛋白內(nèi)部結(jié)構(gòu)展開(kāi)，暴露出抗原表位的原因；當(dāng)加熱溫度在80℃以上，隨著溫度和加熱時(shí)間的增加，β-伴大豆球蛋白α亞基的抗原性降低，這與蛋白質(zhì)二硫鍵的斷裂和重排有關(guān)。當(dāng)加熱溫度為90℃，，處理時(shí)間為35min時(shí)，是β-伴大豆球蛋白α亞基二級(jí)結(jié)構(gòu)被破壞的關(guān)鍵點(diǎn)。 4.將純化得到的α-酪蛋白進(jìn)行脈沖電場(chǎng)處理，在不同處理時(shí)間，處理強(qiáng)度和處理溫度3個(gè)參數(shù)條件下進(jìn)行分析和研究。使用ELISA,圓二色譜，熒光光譜以及紫外光譜等方法研究其抗原性和結(jié)構(gòu)變化之間的關(guān)系。結(jié)果發(fā)現(xiàn)當(dāng)脈沖強(qiáng)度為35kV/cm，處理溫度為10℃以及處理時(shí)間不超過(guò)200μs時(shí)，α-酪蛋白的抗原性降低，而在低強(qiáng)度的脈沖處理以及高溫環(huán)境時(shí)，α-酪蛋白的抗原性都會(huì)升高，這可能是低強(qiáng)度脈沖使蛋白的結(jié)構(gòu)打開(kāi)，暴露了隱藏的抗原結(jié)合位點(diǎn)，高溫能抑制脈沖對(duì)蛋白結(jié)構(gòu)的破壞，而高強(qiáng)度脈沖（35kV/cm）直接破壞其二級(jí)結(jié)構(gòu)。
[Abstract]:Soybean and milk allergies in children and adults in the rate is very high, the milk allergen epitope to understand more clearly, and the sensitization mechanism of soybean beta conglycinin subunit alpha is not completely understood, mainly because it is lack of the allergenic epitopes. Therefore, this paper using DNAStar software, the BepiPred and SOPMA of the three different kinds of biological software to construct epitope prediction method by the prediction of soybean and milk known allergenic epitopes verify the accuracy of the prediction method of original; and this method is successfully applied to the prediction of beta conglycinin subunit epitope, and the predicted epitopes were synthesized, and then use the dot blot to verify the synthetic epitope, get beta conglycinin protein alpha subunit epitope. In addition, the beta conglycinin thermal processing and casein pulse treatment, explore the hot The effects of processing and pulse treatment on the structure and antigenicity of beta globulin and alpha casein, respectively, provide theoretical and technological basis for further development of hypersensitive or anaphylactic soy and dairy products. The main findings and conclusions are as follows.
1. DNAStar, the BepiPred1.0Server, SOPMA Server three bioinformatics software successfully predicted beta conglycinin subunit alpha 15 epitopes. The use of soy allergy serum as antibody, hybridization verified the predicted epitopes, which 11 peptide fragments are thought to be the main source of IgE linear epitope peptide sequence. HEQREEQEWPRKEEKRGEKGSEEEDE is the alpha subunit of the main human IgE epitope. The use of three kinds of software, the predicted epitopes of high accuracy, and provides a new method for prediction of epitopes.
2. to further verify the accuracy of the above method, this method is applied to a known allergen epitope prediction. Using three bioinformatics software DNAStar, SOPMA and the BepiPred Gly m Bd30K on soybean and linear alpha casein epitope prediction, combined with the three level structure of the two kinds of protein Swiss-model a preliminary analysis of 4 epitopes and alpha casein 5 epitope region of Gly m Bd30K. With the known epitopes of the predicted epitopes were compared. Results the correct rate of soybean Gly m Bd30K epitope prediction is 75%, the correct rate of alpha casein was 60%. To lay the foundation for allergen epitope prediction.
3. will be purified with biological activity of beta conglycinin subunit alpha beta by immunizing New Zealand rabbit polyclonal antibody of conglycinin subunit. The polyclonal antibody was applied to ELISA before and after heat treatment, the change detection of beta conglycinin subunit antigen; the round two chromatography, fluorescence spectrum and UV spectrum detection of the structural changes. The results showed that the low temperature (50 DEG -70 DEG) a short time (less than 35min) antigenic heat treatment of beta conglycinin protein alpha subunit is increased, which may be due to the low temperature to the internal structure of protein, epitope exposed reason; when the heating temperature is over 80 DEG C, with the increase of temperature and heating time, reduce the antigenicity of beta conglycinin protein alpha subunit, which is related to the protein two sulfur bond cleavage and rearrangement. When the heating temperature is 90 DEG C, the processing time is 35min, is associated with large beta The key point for the destruction of the two grade structure of the subunit of the bean globulin.
The 4. purified alpha casein by pulsed electric field treatment at different time of treatment, analyze and study the treatment intensity and treatment temperature of 3 parameters. Using ELISA, two circular chromatography, study its antigenicity and structure changes of fluorescence spectra and UV spectra method. The results showed that when the room is 35kV/cm the pulse intensity, processing temperature of 10 DEG C and the processing time does not exceed 200 s, reduce the antigenicity of alpha casein, and in the processing of low intensity pulse and high temperature environment, the antigenicity of alpha casein will increase, which may be of low intensity pulsed protein structure open, exposed the hidden antigen binding site, high temperature can inhibit the pulse of protein structural damage, and high intensity pulse (35kV/cm) direct damage to the secondary structure.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392;TS201.6

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