【作者】 白雪晶；

【导师】 王建华；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 乳铁蛋白是一种天然非血红素铁结合糖蛋白,在哺乳动物非特异性免疫和获得性免疫反应中发挥重要作用。本研究针对连接区域(334-344)结构与功能特点,开展牛乳铁蛋白衍生抗菌片段BLfA、BLfB、BLfN和BLf的分子设计;比较分析了重组蛋白rBLfA、rBLfB、rBLfN和rBLf的Fe3+结合能力和抗菌功能,结果显示牛乳铁蛋白N-叶与C-叶之间α-螺旋连接区域能促进N-叶Fe3+结合结构及功能稳定性,并且为抗菌活性部位;对rBLfA毕赤酵母细胞高密度发酵和代谢工程研究进行了初步探索。主要结果如下:1.利用分子设计方法确定了目标分子BLfA (N-叶和连接区域)及对照组分子BLfB (不完整N-叶)、BLfN (N-叶)和BLf(N-叶、连接区域和C-叶)的一级序列。通过对牛乳铁蛋白分子基本性质的分析和预测,特别是N-叶与C-叶连接区域的结构与功能的分析确立了牛乳铁蛋白衍生抗菌片段的分子设计原则,确定了目标分子BLfA (1-344)及对照组分子BLfB (1-284)、BLfN (1-333)和BLf(1-689)的氨基酸残基序列;利用分子生物学软件对上述分子进行三维结构模拟和物理化学参数计算及对比,结果显示BLfA基本保持了其在天然牛乳铁蛋白中的原有结构,且BLfA分子表面阳离子特性强于BLfB、BLfN和BLf,具有较强的潜在抗菌活性。2.重组表达了牛乳铁蛋白衍生抗菌分子BLfA、BLfB、BLfN和BLf。SDS-PAGE和Western Blotting鉴定显示,BLfA、BLfB和BLf在大肠杆菌BL21(DE3)中以包涵体形式表达,摇瓶水平表达量分别为56 mg/L、41 mg/L和96 mg/L。BLfA、BLfB、BLfN和BLf均在毕赤酵母摇瓶水平得到分泌表达,分别在甲醇诱导30、48、72和72 h后达摇瓶水平最大表达量,依次为485 mg/L、56 mg/L、106 mg/L和88 mg/L。rBLfA、rBLfB、rBLfN和rBLf经Ni-NTA His-Bind树脂纯化后纯度分别为93.8%,92.0%,94.5%和91.2%。3.进行了重组蛋白Fe3+结合及依赖pH的Fe3+释放性质研究,结果表明乳铁蛋白连接区域对N-叶铁离子结合结构的稳定性具有促进作用。Fe3+结合能力nBLf (OD465 nm 0.482)﹥rBLf (0.431)﹥rBLfA (0.330)﹥rBLfN (0.322)﹥rBLfB (0.002),这表明N-叶与C-叶连接区域(334-344)的结构能稳定N-叶的Fe3+结合结构;rBLf与nBLf依赖pH的Fe3+释放性质基本一致,均可在pH 5.5-3.0范围内释放Fe3+,而rBLfA释放Fe3+的pH范围(pH 7.0-3.5)比rBLfN (pH 7.0-4.0)略宽,表明非刚性与刚性的乳铁蛋白连接区结构均可能起到稳定N-叶铁离子结合结构的作用。4.开展了重组蛋白抗金黄色葡萄球菌实验,结果表明牛乳铁蛋白N-叶与C-叶之间的连接区域对其抗菌功能具有促进作用。实验结果显示rBLfA、rBLfN和rBLf均具有抑制S. aureus生长的功能,MIC分别为6.5μmol/L、12. 5μmol/L和25μmol/L,rBLfA抑菌能力最强。rBLfA抑制S. aureus生长活性的增强可能由于其在分子结构上比rBLN多了连接区域α-螺旋,该区域富集的表面净正电荷可能与S. aureus细胞表面带负电荷的分子通过静电相互作用结合,引起细胞裂解,从而起到抑菌和杀菌的作用。5.rBLfA和rBLfN细胞高密度发酵水平的产量分别为499 mg/L和402 mg/L,利用透明颤菌血红蛋白VHb在宿主菌胞内与rBLfA共表达,使rBLfA发酵罐水平的表达量提高了23%,达613 mg/L。更多还原

【Abstract】 Bovine lactoferrin (BLf) is a non-heme iron binding glycoprotein being considered as the first line defense protein involved in protection against microbial infection and prevention of systemic inflammation. The contribution of bovine lactoferrin (BLf) inter-lobe region to iron binding stability and antimicrobial activity against Staphylococcus aureus were addressed in this work.A recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) containing N-lobe (amino acid residues 1-333) and inter lobe region (residues 334-344) of bovine lactoferrin (BLf) was designed based on the structural and functional analysis of BLf. Recombinant full length BLf (rBLf), the N-lobe (rBLfN) without the inter lobe region and the N-lobe-derived peptide rBLfB (residues 1-284) were also designed following the same methords as control. The physical-chemical parameters of BLfA, BLfB, BLfN and BLf including amino acid residues, molecular weight, isoelectric point, net positive charge and instability index were computed and compared. The simulated tertiary structure and the calculated surface net charge showed that rBLfA maintained original structure and exhibited a higher cationic feature than BLfB, BLfN and BLf.The full length cDNA (2067 bp) of BLf and the coding gene of BLfA, BLfB and BLfN were cloned from the bovine mammary tissue. BLfA, BLfB and BLf were expressed in E. coli with the yields of 56, 41 and 96 mg/L, respectively. BLfA, BLfB, BLfN and BLf were all secretory expressed in the methylotrophic yeast Pichia pastoris. The yield of rBLfA in shaking flask culture was over 485 mg/L, which was 4.6, 8.7 and 5.5 times bigger than the yields of rBLfB, rBLfN and rBLf, respectively. Purified rBLfA, rBLfB, rBLfN and rBLf were obtained via Ni-IDA His-bind columns with the purity of 93.8%,92.0%,94.5% and 91.2%.The iron binding and antimicrobial activities comparison between rBLfA, rBLfN and rBLf demonstrates that the inter-lobe region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against S. aureus. The three proteins showed different iron binding stability and antimicrobial activity. rBLfA released iron in the pH range of 7.0-3.5, whereas rBLfN lost its iron over the pH range of 7.0-4.0 and iron release from rBLf occurred in the pH range of 5.5-3.0. However, the minimum inhibition concentration of rBLfA against S. aureus ATCC25923 was 6.5μmol/L, compared with 12.5μmol/L and 25μmol/L that of rBLfN and rBLf, respectively. These results revealed that S. aureus was more sensitive to rBLfA than rBLfN and rBLf. It appeared that the strong cationic character of inter-lobe region related positively to the higher anti-S. aureus activity.High density fermentation and Vitreoscilla hemoglobin (VHb) intracellular co-expression were employed to increase the in vitro expression yield of rBLfA. After 72 h methanol induction, the yield of rBLfA reached 613 mg/L by the high density fermentation of recombinant P. pastoris X-33(rBLfA-VHb).更多还原