增强人α防御素3杀菌效率的研究

发布时间：2018-01-08 18:02

本文关键词：增强人α防御素3杀菌效率的研究　出处：《上海交通大学》2007年硕士论文　论文类型：学位论文

【摘要】： 人α防御素3是由30个氨基酸组成的多肽,富含精氨酸,分子内有3个二硫键。现有研究表明人α防御素3能杀灭革兰氏阳性菌、革兰氏阴性菌、真菌和包被病毒(enveloped virus),但前体蛋白质的杀菌功能不明显。我们用反转录-PCR方法从人白细胞总RNAs扩增出人α防御素3前体的编码序列,克隆到pDEST17表达载体,表达不同结构的人α防御素3蛋白。与前人报道的结果一致,(1)人α防御素3原前体蛋白质(pre-proHNP-31-94),即94个氨基酸组成的多肽,杀菌活性很弱;删除内质网插入的信号肽(第1-19个氨基酸)的α防御素3前体(proHNP-320-94)也不具备杀菌功能;(3)只有成熟的α防御素3(HNP-365-94)能有效地杀灭细菌。非常有兴趣的是,各种构型的人α防御素3,包括原前体蛋白(pre-proHNP-31-94)、前体蛋白质(proHNP-31-94)、以及成熟蛋白质(HNP-365-94)与N-端His6序列的融合都增强了蛋白质的杀菌作用。不能杀菌的原前体和前体能够有效杀菌,其分子当量的杀菌功能弱于没有His6标签的成熟蛋白质。用生物学软件进行分子模拟的结果指出His6序列并不改变蛋白质的整体结构,但能够将蛋白质的等电点向碱性漂移,增加了蛋白质的正电性。这种电荷的改变可能是蛋白质杀菌功能提高的分子基础。基于这些研究,我们认为简单增加碱性氨基酸残基就可能制造出抗菌效率更高防御素。
[Abstract]:Human defensin 3 is a polypeptide composed of 30 amino acid, arginine rich, intramolecular 3 two disulfide bonds. The existing research shows that human alpha defensin 3 can kill gram positive bacteria, gram negative bacteria, fungi and enveloped virus (enveloped virus), but not a precursor protein with bactericidal function obviously. We use the method of reverse transcription -PCR from human leucocyte RNAs amplified the encoding sequence of human defensin 3 precursor, cloned into pDEST17 expression vector and expression of human alpha 3 protein structure in different defense. Results are consistent with previous reports, (1) human alpha defensin 3 pretrypsinogen egg white quality (pre-proHNP-31-94), namely polypeptide of 94 amino acids, the bactericidal activity is very weak; deleted signal peptide inserted into the endoplasmic reticulum (1-19 amino acids) of alpha defensin 3 precursor (proHNP-320-94) does not have the function of sterilization; (3) only 3 mature defense alpha (HNP-365-94) can effectively kill bacteria. Interestingly, human alpha 3 defense in various configurations, including the original precursor protein (pre-proHNP-31-94), precursor protein (proHNP-31-94), and mature protein (HNP-365-94) and fusion N- end His6 sequences both enhanced the bactericidal effect of protein. The original precursor cannot sterilization and precursor can effectively sterilize the molecular equivalent bactericidal function is weaker than the mature protein without His6 tag. The results of molecular simulation were pointed out by biology software His6 sequence does not change the overall structure of the protein, but the isoelectric point of the protein to alkaline drift, increased the positively charged proteins. This charge change may be the molecular basis of protein to improve the sterilization function. Based on these studies, we believe that a simple increase in basic amino acid residues could produce high antimicrobial activity of defensins.

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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