β酪蛋白論文：牛乳中β酪蛋白構(gòu)象性表位定位研究

1、-酪蛋白論文：牛乳中-酪蛋白構(gòu)象性表位定位研究【中文摘要】牛乳是引起食物過敏的八大類常見食物之一。據(jù)報(bào)道,牛乳過敏引發(fā)的病癥在嬰幼兒中尤為常見,1歲以下嬰幼兒中的發(fā)生率約為2-3%。因此,開展牛乳過敏的研究對(duì)于提高牛乳過敏人群生活質(zhì)量具有重要的的現(xiàn)實(shí)意義。另外,由于表位是食物過敏反應(yīng)的物質(zhì)基礎(chǔ),所以表位定位是牛乳過敏研究的一個(gè)核心問題,也是人們認(rèn)識(shí)食物過敏的首要任務(wù)。本文以過敏原蛋白質(zhì)-酪蛋白為研究對(duì)象,從鮮牛乳中分離純化-酪蛋白、制備特異性多克隆抗體、親和純化多克隆抗體、建立-酪蛋白構(gòu)象模型、最終定位出-酪蛋白的構(gòu)象性表位,主要的研究工作和結(jié)論如下。1.采用等電點(diǎn)沉淀法從鮮牛乳中得到酪蛋白,

2、冷凍干燥后的酪蛋白粉末經(jīng)deae-sepharose fast flow陰離子交換柱分離后,得到了-酪蛋白。經(jīng)過sds-page電泳和等電聚焦電泳鑒定,結(jié)果表明,獲得了純度高于90%的-酪蛋白。2.采用分離純化的-酪蛋白免疫日本大白耳兔,制備兔抗牛乳-酪蛋白多克隆抗體,免疫過程中利用間接elisa方法監(jiān)測(cè)免疫效價(jià)的變化,最終得到效價(jià)分別為1：240,000和1：150,000的抗血清各一份。3.利用溴化氰活化的sepharose4b對(duì)抗8-酪蛋白兔血清進(jìn)行親和純化,經(jīng)sds-page電泳檢測(cè),結(jié)果顯示純化后抗體在泳道中基本上只顯示出重鏈(55kda)和輕鏈(22kda)2條帶,純化效果較好；同

3、時(shí)對(duì)純化后抗體進(jìn)行間接elisa效價(jià)測(cè)定,a、b兩兔的血清效價(jià)分別從親和純化上樣前的100,000和70,000上升到純化后的120,000和80,000,表明經(jīng)過親和純化,得到了高純度和高特異性的多克隆抗體。4.分別利用同源建模、折疊識(shí)別法建模、從頭預(yù)測(cè)方法對(duì)-酪蛋白結(jié)構(gòu)模型進(jìn)行構(gòu)建,并對(duì)所建模型進(jìn)行逐級(jí)開放式優(yōu)化和ramanchandran plot、what_check、errat等方法評(píng)估,選擇最優(yōu)模型,結(jié)果得到經(jīng)同源建模并經(jīng)過優(yōu)化后的模型為最終的-酪蛋白結(jié)構(gòu)模型；以文獻(xiàn)報(bào)道的針對(duì)-酪蛋白空間構(gòu)象的實(shí)驗(yàn)數(shù)據(jù)作為參照,將所建模型與kumosinski等人的p-酪蛋白典型模型進(jìn)行比較,評(píng)價(jià)

4、本研究工作中建模結(jié)果的準(zhǔn)確性,結(jié)果顯示,與kumosinski等人所建模型相比,本實(shí)驗(yàn)所建模型更能與-酪蛋白空間構(gòu)象的實(shí)驗(yàn)數(shù)據(jù)吻合。5.通過噬菌體肽庫篩選得到了用于-酪蛋白構(gòu)象性表位定位的6個(gè)模擬肽；同時(shí)通過搜索得到晶體結(jié)構(gòu)已解析的抗原抗體的復(fù)合物,并提取其中的表位信息,形成數(shù)據(jù)信息庫,然后對(duì)該庫進(jìn)行統(tǒng)計(jì)分析,得到表位覆蓋面積大小、表位氨基酸數(shù)目、不同種類表位氨基酸殘基的出現(xiàn)頻率、表位中起間隔作用的氨基酸殘基類型和出現(xiàn)頻率以及表位氨基酸殘基在不同蛋白質(zhì)二級(jí)結(jié)構(gòu)中出現(xiàn)頻率等信息；所得信息結(jié)合噬菌體肽庫篩選結(jié)果,最終定位出-酪蛋白的4個(gè)構(gòu)象性表位。【英文摘要】bovine milk is one

5、of the eight common allergenic foods, it is essential in the different territories and different dietary habits. it is reported that the milk allergy is more prevalent during children, and the incidence in the children aged below 1 year is about 2-3%. undoubtedly, the research on the milk allergy is

6、 important to the improvement of the living quality of the people who suffer from milk allergy. since epitope is the binding sites of antibody and antigen, allergen epitopes will play an important role in food allergens, and epitope mapping should be one of the key problems in the milk allergy resea

7、rch.this work focused on beta-casein, one of the magor allergic proteins in bovine milk, and it includes the purification and separation of the beta-casein, the preparing of specific rabit serum against beta-casein, the purification of the polyclonal antibody by affinity chromatography, modelling an

8、d epitopes mapping of the beta-casein. the detailed process and conclusions are presented as follow.1. crude caseins were prepared from fresh milk using the pi precipitate method, followed by lyophilization, and the dried caseins powder was then loaded into the columns packed with the deae-sepharose

9、 fast flow anion exchange medium. the target eluent then identified by the sds-page and ief page, and it shows the purity of isolated beta-casein was higher than 90%.2. based on the routine way of immunization, specific rbbbit serum against beta-casein was produced. an indirect elisa approach was de

10、veloped to check the change of immune response titer of two rabbits, and the final antiserums titer are 1:240,000 and 1:150,000, respectively.3. the antiserums loaded to sepharose4b which actived by cnbr, the purified components then identified by sds-page, the result illustrated two bands with the

11、molecular weight were 55 kda and 25 kda respectively; also the indirect elisa be carried through to the purified antibody, then it is come to the conclusion that the polyconal antibody with a higher specificity and higher purity;4. the beta-casein models were obtained using the homology modeling met

12、hod, fold recognition and ab initio medhod, the models then were optimized and assessed by ramanchandran plot, what_check, errat and the like, the most optimal model compared with the known model created by kumosinski et al, in this process, the experimental data were used as reference and benchmark

13、, from the results, the conclusion can be drawed that homology model is the most optimal model, and this model is more identical with the experimental data than kumosinski et als model;5.6 mimic peptides obtained after the biopanning process, on the other hand, the antibody-antigen complexes which t

14、he 3d structures were resolved were filtered, then the epitopes information from the complexes were extracted to constitute the epiotpes information database, the information such as the number of the amino acids included in the epitopes, the amino acids preference in the amino acids, the amino acid

15、s which play a interval role in the epitopes, the area the epitopes extend and the like are analysed, finally all these information combined to the pepitides that got from the phage display method for the analysis of epitopes, then four conformational epitopes were obtained in this research.【關(guān)鍵詞】-酪蛋

16、白 同源建模 表位 牛乳過敏【英文關(guān)鍵詞】-casein homology modeling epitope bovine milk allergy【目錄】牛乳中-酪蛋白構(gòu)象性表位定位研究摘要3-5abstract5-6目錄9-14第1章 引言14-261.1 牛乳過敏14-171.1.1 牛乳過敏機(jī)理15-161.1.2 牛乳中的主要過敏原161.1.3 牛乳過敏的危害16-171.2 牛乳過敏原的安全管理17-201.2.1 牛乳過敏的檢測(cè)17-191.2.1.1 血液檢測(cè)17-181.2.1.2 血液檢測(cè)181.2.1.3 食物激發(fā)試驗(yàn)18-191.2.2 牛乳過敏原的管理19-201.

17、2.2.1 ige介導(dǎo)的牛乳過敏的管理191.2.2.2 非ige介導(dǎo)的牛乳過敏的管理191.2.2.3 針對(duì)不同過敏癥狀的牛乳過敏的管理191.2.2.4 食物避免與營養(yǎng)的管理19-201.2.2.5 食物過敏原標(biāo)簽管理201.3 牛乳中的酪蛋白20-231.3.1 酪蛋白膠束20-211.3.2 牛奶酪蛋白的結(jié)構(gòu)與理化性質(zhì)21-231.3.2.1 s1-酪蛋白21-221.3.2.2 _(s2)-酪蛋白221.3.2.3 -酪蛋白22-231.3.2.4 -酪蛋白231.4 -酪蛋白的構(gòu)象性表位23-241.5 研究內(nèi)容及意義24-26第2章 牛乳中-酪蛋白的分離純化26-352.1 材料

18、與設(shè)備26-272.1.1 試劑與材料262.1.2 設(shè)備26-272.1.3 溶液配制272.2 實(shí)驗(yàn)方法27-302.2.1 鮮牛奶脫脂272.2.2 等電點(diǎn)沉淀酪蛋白272.2.3 -酪蛋白預(yù)處理27-282.2.4 -酪蛋白分離純化282.2.5 蛋白質(zhì)及其純度鑒定28-292.2.5.1 sds-page鑒定28-292.2.5.2 ief-page鑒定292.2.6 蛋白質(zhì)濃度鑒定29-302.3 實(shí)驗(yàn)結(jié)果30-332.3.1 -酪蛋白的分離純化30-312.3.2 蛋白質(zhì)及其純度鑒定31-322.3.3 濃度鑒定32-332.4 討論33-342.4.1 -酪蛋白的離子交換層析條

19、件選擇332.4.2 蛋白質(zhì)鑒定和濃度鑒定33-342.5 小結(jié)34-35第3章 抗牛乳-酪蛋白多克隆抗體的制備及親和純化35-483.1 材料與設(shè)備35-373.1.1 試劑353.1.2 儀器與耗材35-363.1.3 溶液配制36-373.2 實(shí)驗(yàn)方法37-403.2.1 多克隆抗體的制備373.2.2 抗體滴度測(cè)定的方正滴定373.2.3 間接elisa測(cè)定抗體效價(jià)37-383.2.4 抗血清的制備383.2.5 親和層析純化特異性兔igg38-403.2.5.1 血清的預(yù)處理383.2.5.2 親和層析柱的制備38-393.2.5.3 親和純化393.2.5.4 抗體sds-page

20、純度鑒定393.2.5.5 間接elisa測(cè)定抗體效價(jià)39-403.3 實(shí)驗(yàn)結(jié)果40-443.3.1 elisa方法的條件確立及其對(duì)效價(jià)變化的監(jiān)測(cè)40-413.3.2 親和層析純化特異性igg41-443.3.2.1 血清的獲取413.3.2.2 偶聯(lián)效果的檢測(cè)413.3.2.3 非特異性洗脫過程中蛋白質(zhì)含量監(jiān)測(cè)41-423.3.2.4 特異性洗脫過程中蛋白質(zhì)含量監(jiān)測(cè)42-433.3.2.5 濃縮除鹽433.3.2.6 純化前后抗體效價(jià)的比較433.3.2.7 純化后樣品的sds-page電泳圖43-443.4 討論44-473.4.1 佐劑的選擇44-453.4.2 試驗(yàn)動(dòng)物的選擇453.4.3 多克隆抗體的制備和儲(chǔ)存45-463.4.4 多克隆抗體的親和純化46-473.4.4.1 配基及載體的選擇463.4.4.2 層析條件的選擇46-473.5 本章小結(jié)47-48第4章 -酪蛋白結(jié)構(gòu)模型的構(gòu)建48-584.1 實(shí)驗(yàn)材料484.2 實(shí)驗(yàn)原理和方法48-524.2.1 蛋白質(zhì)結(jié)