大豆和牛奶致敏原表位预测及加工对其抗原性和结构的影响

发布时间：2018-01-12 00:02

本文关键词：大豆和牛奶致敏原表位预测及加工对其抗原性和结构的影响　出处：《江南大学》2013年硕士论文　论文类型：学位论文

【摘要】：大豆和牛奶过敏在儿童和成年人中的发病率都很高，目前对于牛奶致敏原的表位了解的比较清楚，而对大豆β-伴大豆球蛋白α亚基的致敏机理尚不完全清楚，主要因为是缺乏对其致敏表位的认识。因此，本论文借助DNAStar软件，the BepiPred和SOPMA这三种不同的生物软件构建表位预测方法，通过预测大豆和牛奶已知表位的致敏原来验证预测方法的准确性；并将该方法成功应用于预测β-伴大豆球蛋白α亚基表位，并对预测得到的表位进行合成，然后使用点杂交方法对合成的表位进行验证，得到β-伴大豆球蛋白α亚基的表位。另外，通过对β-伴大豆球蛋白热加工和α-酪蛋白脉冲处理，探讨热加工处理和脉冲处理分别对β-伴大豆球蛋白和α-酪蛋白结构和抗原性的影响，为进一步开展低过敏性或无过敏性的大豆和牛奶制品加工提供理论和技术依据，主要研究结果及结论如下： 1. DNAStar，the BepiPred1.0Server，SOPMA网络服务器三种生物信息学软件成功预测得到β-伴大豆球蛋白α亚基的15段表位。使用人大豆过敏血清作为抗体，点杂交技术验证预测得到的表位，其中11段肽片段被认为是主要人源IgE线性表位。肽序列HEQREEQEWPRKEEKRGEKGSEEEDE是α亚基最主要的人源IgE表位。使用三种软件预测得到的表位准确率较高，为表位的预测提供一种新方法。 2.为进一步验证上述方法的准确性，将该方法应用于已知表位的过敏原的预测。使用三种生物信息学软件DNAStar，SOPMA和the BepiPred对大豆Gly m Bd30K以及α-酪蛋白的线性表位进行预测，同时结合Swiss-model对这两种蛋白的三级结构进行了初步分析，得到Gly m Bd30K的4个表位区域以及α-酪蛋白的5个表位区域。将预测得到的表位与已知的表位进行了比较，结果大豆Gly m Bd30K预测表位的正确率为75%，α-酪蛋白的正确率为60%，为致敏原表位预测奠定基础。 3.将纯化得到具有生物活性的β-伴大豆球蛋白α亚基免疫新西兰大白兔得到β-伴大豆球蛋白α亚基的多克隆抗体。将多克隆抗体应用于ELISA，检测热处理前后β-伴大豆球蛋白α亚基抗原性的变化；使用圆二色谱，荧光光谱以及紫外光谱检测其结构变化。结果发现低温（50℃-70℃）短时间（35min以内）加热处理β-伴大豆球蛋白α亚基的抗原性是升高的，这可能是由于低温使蛋白内部结构展开，暴露出抗原表位的原因；当加热温度在80℃以上，随着温度和加热时间的增加，β-伴大豆球蛋白α亚基的抗原性降低，这与蛋白质二硫键的断裂和重排有关。当加热温度为90℃，，处理时间为35min时，是β-伴大豆球蛋白α亚基二级结构被破坏的关键点。 4.将纯化得到的α-酪蛋白进行脉冲电场处理，在不同处理时间，处理强度和处理温度3个参数条件下进行分析和研究。使用ELISA,圆二色谱，荧光光谱以及紫外光谱等方法研究其抗原性和结构变化之间的关系。结果发现当脉冲强度为35kV/cm，处理温度为10℃以及处理时间不超过200μs时，α-酪蛋白的抗原性降低，而在低强度的脉冲处理以及高温环境时，α-酪蛋白的抗原性都会升高，这可能是低强度脉冲使蛋白的结构打开，暴露了隐藏的抗原结合位点，高温能抑制脉冲对蛋白结构的破坏，而高强度脉冲（35kV/cm）直接破坏其二级结构。
[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.

【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392;TS201.6

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