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1、上海交通大学 硕士学位论文 乳铁蛋白的仿生亲和纯化研究 姓名：文胜 申请学位级别：硕士 专业：微生物 指导教师：李荣秀 20090101 上海交通大学硕士研究生学位论文 - I - 乳铁蛋白的仿生亲和纯化研究乳铁蛋白的仿生亲和纯化研究 摘 要 摘 要 人乳铁蛋白是一种分子质量为 7080 kD 的糖蛋白，属转铁蛋白家 族成员，具有广谱抗菌，抗病毒，抗肿瘤，调节机体免疫反应等多种生 物活性，在功能营养食品和医疗保健等方面具有广阔的应用前景。过去， 因人乳铁蛋白来源有限，严重限制了其商业开发。随着动物转基因技术 的发展，中国农业大学李宁教授研究团队构建了牛乳腺生物反应器，在 牛乳腺中高量表达重组人
2、乳铁蛋白，牛乳中人乳铁蛋白浓度达 2.529 0.212 mg/mL3.4290.417 mg/mL，有可能实现重组人乳铁蛋白在牛乳 腺中的规模生产。本课题研究目的是针对乳铁蛋白研究开发仿生亲和配 体，仿生亲和分离材料以及建立从转基因牛乳中大规模纯化重组人乳铁 蛋白的工艺。 本研究首先设计合成了一个仿生亲和分离材料库，含322中不同结构 的配体；利用筛选方法得到了特异性结合重组人乳铁蛋白的-丙氨酸亲 和配体，其结构为-丙氨酸经三氯三嗪间隔臂共价偶联于氨基活化的 Sepharose-6B上。通过对固定化-丙氨酸纯化重组人乳铁蛋白的pH和离 子强度条件进行优化，确定介质对重组人乳铁蛋白的最佳结合条
3、件为： 50 mM Tris-HCl/0.1 M NaCl, pH 7.0； 最佳洗脱条件为： 0.1 M Gly-HCl, pH 2.4。纯化得到的重组人乳铁蛋白纯度95%，回收率达81.6%，固定化- 丙氨酸对重组人乳铁蛋白的最大吸附量达65.6mg/g 干介质，并且能够耐 受NaOH溶液的清洗与再生。 经MALDI-TOF鉴定，所纯化的蛋白即为重组人乳铁蛋白。N端测序 结果表明，重组人乳铁蛋白N端氨基酸序列为GRRRRSVQW，与天然人 乳铁蛋白相同。 上海交通大学硕士研究生学位论文 - II - 对重组人乳铁蛋白最重要的两个生物学活性进行了鉴定。在体外经 铁溶液处理后，重组与天然人乳铁
4、蛋白均能结合铁离子，而且这种铁结 合能力与环境中的pH直接相关。当pH降至4.5时，铁离子开始从蛋白中释 放出来。在pH 2.0时，蛋白几乎全部释放掉自身结合的铁离子。这表明重 组人乳铁蛋白具有与天然人乳铁蛋白类似的铁结合与释放能力。重组人 乳铁蛋白具有抑菌活性，当大肠杆菌菌浓度为1105CFU/mL时，加入终 浓度为5 mg/mL的重组人乳铁蛋白能明显抑制细菌的生长。 除重组人乳铁蛋白外，固定化-丙氨酸可从人乳，天然牛乳和羊乳 中纯化乳铁蛋白，表明该介质可用于不同种属乳铁蛋白的亲和纯化。 以上结果表明，利用固定化-丙氨酸亲和介质可以大规模纯化具有 天然生物学活性的重组人乳铁蛋白，并且可以应用
5、于从人乳，天然牛乳 和羊乳等乳汁中纯化不同种属的乳铁蛋白。 关键词：关键词：转基因牛乳，重组人乳铁蛋白，仿生亲和配体，亲和纯化 上海交通大学硕士研究生学位论文 - III - Affinity Purification of Lactoferrin by Synthetic ligand ABSTRACT Human lactoferrin is a 7080 kDa ironbinding glycoprotein of the transferring family with antibacterial, antifungal, antiviral, antitumor and immun
6、omodulating activities. Owing to its implications in these functions, human lactoferrin has been considered a wide variety of applications in food and human health care, but its applications are restricted because of the limited source. Recently, a cattle mammary gland bioreactor expressing recombin
7、ant human lactoferrin has been generated by a research group led by Dr. Li Ning at Chinese Agricultural University, the level of recombinant human lactoferrin expressed by transgenic cattle could reach as high as 2.529 0.212 mg/mL3.4290.417 mg/mL, which probably provided a powerful approach to achie
8、ve large-scale production of recombinant human lactoferin in the milk of transgenic cows. The purpose of this study is to establish a novel affinity protocol for large-scale purification of recombinant human lactoferrin from the milk of transgenic cows by synthetic ligands. Immobilized -Alanine, a s
9、ynthetic ligand with high affinity for recombinant human lactoferrin was selected based on screening of a 322-member synthetic ligand library. By further study, TrisHCl buffer(50 mmol/L, pH 7.0, 0.15 mol/L NaCl) and GlyHCl buffer(0.1 mol/L, pH 2.4) 上海交通大学硕士研究生学位论文 - IV - were chosen as equilibration
10、 buffer and elution buffer respectively for this affinity protocol. By one-step purification with immobilized -Alanine, the bound recombinant human lactoferrin was eluted with purity95%, and the recovery could reach 81.6%. The maximal binding capacity of immobilized -Alanine for recombinant human la
11、ctoferrin is as high as 65.5 mg/ g dry medium, moreover, this synthetic ligand could withstand the harsh cleaning and regeneration by NaOH solution. MALDI-TOF analysis identified that the purified protein was human lactoferrin, and the N-terminal sequencing further revealed that the N-terminal seque
12、nce of recombinant human lactoferrin was identical to that of native human lactoferrin, i.e., GRRRRSVQW. Tow most prominent biological activities of human lactoferrin, that is, iron-binding and releasing properties and antibacterial activity, were investigated, and the results indicated that the bio
13、logical activities of recombinant human lactoferrin were similar to that of native human lactoferrin. Both the recombinant human lactoferrin and native human lactoferrin could bind iron after treatment with Fe-NTA solution and release iron when pH was low. The iron began to be released around pH 4.5
14、 and completely finished around pH 2.0. The growth of E.coli. was significantly inhibited when 5 mg/mL recombinant human lactoferrin was added to bacterial medium, which revealed that the recombinant human lactoferrin retained its natural biological activity after undergoing purifying procedures. 上海
15、交通大学硕士研究生学位论文 - V - In additional, immobilized -Alanine could purify native human lactoferrin, bovine lactoferrin and goat lactoferrin from human milk, bovine milk and goat milk respectively, which predicts a vast potential for multiple applications of this ligand. In conclusion, immobilized -Alanin
16、e can be applied to large-scale purification of recombinant human lactoferrin as well as native human lactoferin, bovine lactoferrin and goat lactoferrin respectively and has great potential for commercial application. Keywords: milk of transgenic cows, recombinant human lactoferrin, synthetic ligand, affinity purification 附件四附件四 上海交通大学上海交通大学 学位论文原创性声明学位论文原创性声明 本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独 立进行研究工作所取得的成果。除文中已经注明引用的内容外，本论 文不包含任何其他个人或集体已经发表或撰写过的作品成果。对本文 的研究做出重要贡献的个人和集体
