本文選題：蝦過(guò)敏原 + 鑒定��； 參考：《中國(guó)海洋大學(xué)》2011年碩士論文

【摘要】：隨著現(xiàn)代社會(huì)的發(fā)展,人們飲食結(jié)構(gòu)的變化,與食物直接相關(guān)的過(guò)敏疾病發(fā)病率日益增高,給人們的生命健康造成了巨大威脅。蝦類(lèi)產(chǎn)品屬于我國(guó)產(chǎn)值比較高的甲殼類(lèi)海產(chǎn)品,由于味道鮮美、營(yíng)養(yǎng)豐富,深受消費(fèi)者的喜愛(ài)。然而蝦類(lèi)海產(chǎn)品屬于8大類(lèi)食物過(guò)敏原之一,使許多消費(fèi)者飽受過(guò)敏癥狀的困擾。本論文著重對(duì)蝦類(lèi)過(guò)敏原的鑒定和活性消除方法進(jìn)行研究,結(jié)合生物信息學(xué)方法進(jìn)一步分析過(guò)敏原的表位區(qū)域及氨基酸組成性質(zhì),并對(duì)表位進(jìn)行空間定位。主要內(nèi)容如下: 1、以刀額新對(duì)蝦為研究對(duì)象,分離和純化了分子量為36kD的主要過(guò)敏原蛋白,利用基質(zhì)輔助激光解析電離/飛行時(shí)間質(zhì)譜對(duì)其進(jìn)行肽質(zhì)量指紋圖譜鑒定,并對(duì)得到的結(jié)果采用Mascot搜索引擎在NCBInr數(shù)據(jù)庫(kù)上進(jìn)行搜索。結(jié)果表明刀額新對(duì)蝦主要過(guò)敏原為原肌球蛋白,與斑節(jié)對(duì)蝦原肌球蛋白匹配分值最高為268,吻合肽段27條,序列覆蓋率為65%;與其它無(wú)脊椎動(dòng)物如腐食酪螨、衣魚(yú)等的原肌球蛋白的序列覆蓋率也很高。這一結(jié)果不僅表明了刀額新對(duì)蝦與其它甲殼類(lèi)海產(chǎn)品過(guò)敏原存在著高度同源性,而且為其與甲殼類(lèi)及其它無(wú)脊椎動(dòng)物主要過(guò)敏原之間存在嚴(yán)重交叉反應(yīng)的現(xiàn)象提供了理論依據(jù)。 2、在鑒定了刀額新對(duì)蝦的主要過(guò)敏原之后,進(jìn)一步分析了常見(jiàn)熱加工方式對(duì)其過(guò)敏原活性的影響。選取煮、蒸、高壓這三種方法,分別處理刀額新對(duì)蝦不同時(shí)間。通過(guò)檢測(cè)總蛋白和主要過(guò)敏原含量以及活性的變化,分析對(duì)蝦類(lèi)過(guò)敏原的影響。結(jié)果表明經(jīng)過(guò)三種熱處理后,36kD主要過(guò)敏原蛋白仍然存在,但其活性有不同程度的下降。在25-35kD區(qū)域出現(xiàn)一條新的免疫活性蛋白,但影響不大,全蛋白的免疫活性仍有不同程度的降低。其中高壓對(duì)活性的降低程度最大,30分鐘時(shí)免疫活性降低了97%。但質(zhì)地剖面分析結(jié)果表明,高壓處理對(duì)蝦肉質(zhì)構(gòu)破壞最大,仍需進(jìn)一步優(yōu)化高壓處理工藝,在降低蝦致敏活性的同時(shí),保持其口感及營(yíng)養(yǎng)。 3、利用生物信息學(xué)方法對(duì)蝦過(guò)敏原及其他甲殼類(lèi)過(guò)敏原之間的同源性進(jìn)行分析,并繪制出系統(tǒng)進(jìn)化樹(shù)。結(jié)果表明甲殼類(lèi)過(guò)敏原原肌球蛋白氨基酸序列十分保守,序列之間同源性很高。其中斑節(jié)對(duì)蝦、凡納濱對(duì)蝦過(guò)敏原同褐美對(duì)蝦主要過(guò)敏原Pen a 1的氨基酸序列完全相同。 采用DNAstar、AntheProt等生物信息學(xué)軟件分析Pen a 1的二級(jí)結(jié)構(gòu)、親水性、溶劑可及性、可塑性、抗原指數(shù)等多個(gè)性質(zhì),間接預(yù)測(cè)線性表位區(qū)域,并結(jié)合2個(gè)在線網(wǎng)站對(duì)表位的預(yù)測(cè)結(jié)果進(jìn)行篩選,得到10個(gè)抗原表位后進(jìn)行表位肽的固相合成。采用競(jìng)爭(zhēng)Dot-blot方法,利用患者血清對(duì)表位肽的活性進(jìn)行初步驗(yàn)證。結(jié)果表明其中8個(gè)表位多肽為主要過(guò)敏原表位,其中有兩個(gè)以前未曾有研究者報(bào)道過(guò)。對(duì)表位區(qū)域的氨基酸分析中發(fā)現(xiàn)精氨酸(R)、酪氨酸(Y)、苯丙氨酸(F)、絲氨酸(S)以及谷氨酸(E)這5種氨基酸在表位區(qū)域出現(xiàn)概率很高。 4、以豬原肌球蛋白1c1gC為模板,利用SWISS-MODEL以及CPHmodels中相關(guān)的同源建模功能對(duì)Pen a 1的空間結(jié)構(gòu)進(jìn)行預(yù)測(cè)。采用原子平均勢(shì)能、分子體系動(dòng)力學(xué)分析以及拉氏構(gòu)象圖對(duì)建模結(jié)果進(jìn)行評(píng)估。結(jié)果表明模擬形成的Pen a 1構(gòu)象有較高的穩(wěn)定性和合理性。從構(gòu)象中可以看出, Pen a 1空間結(jié)構(gòu)比較簡(jiǎn)單,沒(méi)有復(fù)雜的三、四級(jí)結(jié)構(gòu),二級(jí)結(jié)構(gòu)主要以α-螺旋為主。從構(gòu)象方面分析,Pen a 1單鏈自身形成構(gòu)象型表位的幾率不大,而線性表位在空間中的定位結(jié)果也顯示基本上線性表位全部暴露在外。 5、利用氨基酸突變的方法研究Pen a 1表位中的關(guān)鍵氨基酸,選擇Pen a 1中peptide6和peptide10這兩條表位肽,將其中的活性氨基酸替代為人或豬原肌球蛋白中相應(yīng)的非活性氨基酸。采用Dot-blot競(jìng)爭(zhēng)酶聯(lián)免疫的方法檢測(cè)突變表位肽過(guò)敏活性的變化,分析對(duì)活性影響較大的氨基酸殘基。結(jié)果表明,Pen a 1中Peptide 10中所對(duì)應(yīng)的表位區(qū)域中,278F和279S氨基酸替代為人原肌球蛋白相應(yīng)位置的氨基酸L后,突變表位肽與過(guò)敏患者血清IgE的結(jié)合能力有明顯的下降,說(shuō)明這兩者為該表位中的關(guān)鍵氨基酸。其他表位中的關(guān)鍵氨基酸還有待于進(jìn)一步分析。
[Abstract]:With the development of modern society, the change of people's diet structure, the incidence of allergic diseases which are directly related to food is increasing, which poses a great threat to people's life and health. Shrimp products belong to the high value crustaceans of our country, which are delicious, rich in camping and are loved by consumers. However, shrimp seafood is a kind of seafood. It is one of the 8 major food allergen, which makes many consumers plagued by allergy symptoms. This paper focuses on the identification and activity elimination methods of shrimp allergen, and further analyzes the surface and amino acid composition of allergens with bioinformatics methods, and makes spatial location of the epitopes. The main contents are as follows:
1, the main allergen protein with molecular weight of 36kD was isolated and purified, and the peptide mass fingerprint was identified by matrix assisted laser analytical ionization / time of flight mass spectrometry, and the results were searched by the Mascot search engine on the NCBInr database. The results showed that the knife forehead was the new prawn owner. The anaphylaxis was original myosin, and the maximum matching score of the myosin of Penaeus Penaeus was 268, the peptide segment was 27, the sequence coverage was 65%, and the sequence coverage of the myosin of other invertebrates, such as the chitin mite and the coat fish, was also high. This result not only indicated the allergens of the new shrimp and other crustaceans in the shrimp and other crustaceans. There is a high degree of homology and provides a theoretical basis for the severe cross reaction between them and crustaceans and other invertebrates.
2, after identifying the main allergen of Penaeus Penaeus, the influence of the common hot processing methods on the activity of allergens was further analyzed. The three methods of cooking, steaming and high pressure were selected to deal with the different time of the new prawns. The changes of the total protein, the main allergens and the activity of the allergens were detected, and the shadow of the shrimp allergen was analyzed. The results showed that after three kinds of heat treatment, the main allergen protein of 36kD still existed, but its activity decreased in varying degrees. There was a new immuno active protein in the 25-35kD region, but the effect of the whole protein was still reduced in varying degrees. Among them, the activity was most reduced by high pressure, and the immunity was 30 minutes. The activity of 97%. was reduced, but the texture profile analysis showed that high pressure treatment of shrimp meat texture was the greatest damage. It still needed to further optimize the high pressure treatment process, while reducing the sensitization activity of shrimp, maintaining its taste and nutrition.
3, using bioinformatics method to analyze the homology of shrimp allergen and other crustacean allergens, and draw a phylogenetic tree. The results show that the amino acid sequence of the crustacean allerogen myosin amino acid is very conservative and the sequence is very homologous. The amino acid sequence of the Pen a 1 is exactly the same.
DNAstar, AntheProt and other bioinformatics software are used to analyze the two grade structure of Pen a 1, hydrophilicity, solvent accessibility, plasticity and antigen index, and indirectly predict the linear epitope region, and combine 2 online sites to screen the prediction results of the epitopes and obtain the solid phase synthesis of epitopes after 10 epitopes. The Dot-blot method was used to verify the activity of the epitope of the patient's sera. The results showed that 8 epitopes were the main allergenic epitopes, and two of them had not been reported before. The amino acid analysis of the epitopes found that arginine (R), tyrosine (Y), phenylalanine (F), serine (S), and glutamic acid (E) were found. The probability of the 5 amino acids in the epitope region is very high.
4, using the porcine proomyosin 1c1gC as a template, the spatial structure of Pen a 1 was predicted by using the related homologous modeling functions of SWISS-MODEL and CPHmodels. The results were evaluated by atomic mean potential energy, molecular dynamics analysis and Lagrangian conformation diagram. The results showed that the conformation of the Pen a 1 formed by the simulation had high stability. It can be seen from the conformation that the spatial structure of Pen a 1 is simple, there is no complex three, four structure, and the two structure is mainly alpha helix. From the conformation analysis, the probability of forming conformational epitopes of the single strand of Pen a 1 is not good, and the linear epitope in the space also shows the basic linear epitopes. Exposure to the outside.
5, the key amino acids in the Pen a 1 epitopes were studied by amino acid mutation, and two epitopes of peptide6 and peptide10 in Pen a 1 were selected to substitute the active amino acids in the human or porcine promyosin as the corresponding inactive amino acids. The mutation of the mutant epitopes was detected by the Dot-blot competitive enzyme immunoassay. The results showed that in the epitope region corresponding to the Peptide 10 in Pen a 1, 278F and 279S amino acids were substituted for the corresponding amino acid L of human myosin, and the binding ability of the mutant epitopes to the serum IgE in the allergic patients was significantly decreased, indicating that both of these were the key points in the epitope. Key amino acids. Other key amino acids in other epitopes need further analysis.
【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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