【作者】 郭峰；

【导师】 高剑峰； 王慧；

【作者基本信息】 石河子大学 ， 遗传学， 2013， 硕士

【摘要】 目的：以人β防御素（hBDs）为基础，通过改变特定氨基酸序列获得原防御素的突变体，旨在提高其杀菌活力，同时降低盐敏感性，并初步探讨防御素的构效关系与机理。摸索防御素的原核表达途径。方法：（1）Fmoc法合成防御素，对合成后的防御素进行初步的纯度分析以及结构分析。（2）对合成的防御素进行杀菌以及盐敏感性的分析，并应用透射电镜观察防御素的作用方式。（3）用MTT法对合成防御素进行细胞毒性分析，同时通过溶血实验来分析其对血细胞的毒性。（4）将hBD-3进行原核表达纯化。结果：（1）在人β防御素1-4（hBD-1-4）中，hBD-3对测试菌有最强的杀菌活力及最强的耐盐活力，对测试菌株的百分之九十杀死率（LD90）在15μg/ml以下。同时，合成的hBD-1抑菌活力很低，LD90超过100μg/ml。合成的hBD-2,4抑菌活力弱于hBD-3（P＜0.01），但并没有发现明显的对革兰氏阴阳菌的选择性。（2）hBD-3的N端缺失突变体当失去前9个氨基酸时活性及耐盐能力显著下降（P＜0.01），而失去前3个氨基酸的突变体耐盐能力显著提高（P＜0.01）。（3）经过I区替换的改构防御素都有着比hBD-3更强的杀菌活力及耐盐能力，而它们各自的N端缺失突变体杀菌活性及耐盐能力降低。（4）改构体在耐盐能力提高的同时，它们的细胞毒性以及溶血毒性也相应提高。（5）电镜结果显示，防御素作用于细菌后，破坏细胞壁的同时造成质壁分离，导致细菌死亡。（6）成功构建了原核表达载体表达出融合蛋白GST-hBD-3。结论：（1）在hBD1-4中，hBD-3的活性，耐盐能力在所测试的菌株中最强，可以将它作为改构的模板。（2）hBD-3的N端区域对防御素的盐敏感性有着重要作用。（3）hBD-3的I区对防御素杀菌活力有着重要影响。而对于防御素杀菌活性与细胞毒性的关系，需要在后期实验中进一步研究。更多还原

【Abstract】 Object: Chose Human beta-defensin1-4(hBD-1-4) as template, to design new defensin analogs. Aimed atimproving their antibacterial activity, while reducing salt sensitivity and toxicity of eukaryotic cells, andmade a speculation of structure-activity relationships and mechanism of defensins. Attempt the way to getprotein from prokaryotic cell protein expression method.Methods:(1) Defensins were synthesized by Fmoc method, and analysed their purity and structureprimarily.(2) Analysed defensins’ antibacterial activity and salt sensitivity, and observed their role ofkilling bacteria by transmission electron microscopy.(3) Mensurated the cytotoxicity (MTT method) andhymolysis of beta-defensins and its analogs.(4) Construct a expression vector to express hBD-3.Results:(1) HBD-3has the strongest antibacterial activity and salt resistant ability among hBD-1-4, itsLD90s to the bacteria in this experiment were lower than15μg/ml. At the same time, the LD90s of hBD-1were higher than100μg/ml. The antimicrobial activity of human hBD-2, hBD-4were weaker than hBD-3(P＜0.01).(2) The three N-terminal amino acid analog was more salt resistant than hBD-3while itscytotoxicity became stronger obviously, while the antimicrobacterial activity of the nine N-terminal aminoacid analog was weaker than hBD-3.(3) The I region changed analogs analogs had a stronger antibacterialactivity and salt resistant ability than hBD-3, but these ability of their N-terminal deletion analogs becameweaker.(4) We found that the analogs which had a higher salt resistant ability were always more toxic.(5)Transmission electron microscopy showed, defensin destoryed cell wall and cell membrane, and then leada death of bacteria.(6) We sucessfully expressed a fusion protein GST-hBD-3.Conclusion:(1) It is a short cut that using hBD-3as a template in defensin reconstruction.(2) TheN-terminal amino acid is important for salt sensitivity.(3) The I region of hBD-3is important for itsantibacterial activity. In later work, we will study the relationship between antibacterial activity andcytotoxicity of defensin.更多还原