重组嵌合抗菌多肽的构建及其在抗感染和抗肿瘤免疫中的作用

发布时间：2018-03-11 18:10

本文选题：嵌合蛋白　切入点：抗菌肽　出处：《四川大学》2005年博士论文　论文类型：学位论文

【摘要】：细菌及病毒感染是困扰人类很久的问题,尤其是近年来由于抗生素不规范使用及细菌突变等多方面原因,耐药菌的感染成为日益严重而急需解决的一个难题。内源性抗菌肽是一类阳离子短肽,广泛分布于人体多个部位,具有广谱抗微生物活性,在不久的将来可能作为新一代抗感染药物代替传统抗生素。防御素是人体中一类重要抗菌肽,新近的研究发现该类分子还是一种多功能免疫活性肽,广泛地参与了机体的免疫应答。本研究应用重组嵌合蛋白技术试图改造抗菌肽以提高抗菌活性和稳定性,构建抗菌肽嵌合核酸疫苗以增强抗肿瘤免疫。本文分三个部分。第一部分是对本实验室从人子宫颈粘液中分离鉴定出的1个新抗菌肽进行N-端氨基酸序列测定和cDNA克隆,获得其全长编码基因,以便用作嵌合肽的构建。测序结果显示氮端氨基酸序列从第一至第十位依次为脯氨酸(Pro,P),赖氨酸(Lys,K),精氨酸(Arg,R),赖氨酸(Lys,K),丙氨酸(Ala,A),天冬氨酸(Asp,D),甘氨酸(Gly,G),谷氨酸(Glu,E),丙氨酸(Ala,A),赖氨酸(Lys,K)。根据其氮端氨基酸序列,设计简并引物,3’-RACE-PCR技术扩增出全长cDNA,通过BLAST程序检索对比分析,确定其为高迁移率蛋白N2(High mobility group chromosomal protein N2, HMG N2),用蛋白质二结构软件分析发现该分子含1个跨膜α-螺旋结构域,位于该分子氨基酸序列的第17位至42位之间。化学合成该分子N-端、α-螺旋和C-端肽片段,抗菌活性检测显示仅α-螺旋肽片段具有抗菌活性,证明α-螺旋是HMG N2抗菌功能结构域。第二部分是构建HMG N2 α-螺旋结构域与富组蛋白-5嵌合抗菌多肽。采用PCR拼接技术将二者连接起来,构建出融合肽原核表达质粒
[Abstract]:Bacterial and viral infections have been a problem for a long time, especially due to the irregular use of antibiotics and bacterial mutations in recent years. The infection of drug-resistant bacteria has become an increasingly serious and urgent problem. Endogenous antimicrobial peptides are a kind of cationic short peptides widely distributed in many parts of human body and have broad spectrum antimicrobial activity. In the near future, defensins may replace traditional antibiotics as a new generation of anti-infective drugs. Defensins are an important class of antimicrobial peptides in the human body, and recent studies have found that these molecules are also a multifunctional immunoreactive peptide. In this study, recombinant chimeric protein technology was used to modify antimicrobial peptides to improve their antibacterial activity and stability, and to construct an antimicrobial peptide chimeric nucleic acid vaccine to enhance anti-tumor immunity. This paper is divided into three parts. In the first part, a new antimicrobial peptide isolated from human cervical mucus in our laboratory was sequenced by N-terminal amino acid and cloned into cDNA, and its full-length encoding gene was obtained. Sequence analysis showed that the N-terminal amino acid sequences from the first to the 10th were proline, lysine, arginine, Alaanine, aspartic acid, glycine, glutamate, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamate, glutamate, glutamate, glutamate, glutamic acid, glutamate, glutamic acid, glutamate, glutamate, glutamic acid, glutamate, glutamate, glutamic acid, glutamic acid, and alanine. The amino acid sequence of the nitrogen terminal of Alaa, Lyschon, and its amino acid sequence was determined by analyzing the amino acid sequence of the amino acid terminal of the amino acid. The full-length cDNA was amplified by degenerate primer 3Case -RACE-PCR, and was identified as high mobility protein N2 high mobility group chromosomal protein N2, HMG N2 by BLAST program search and contrast analysis. A transmembrane 伪 -helix domain was found by protein two-structure software analysis. The N-terminal, 伪 -helix and C-terminal peptide fragments were chemically synthesized from the 17th to the 42nd position of the amino acid sequence of the molecule. The antibacterial activity of 伪 -helix peptide fragment was only found to have antibacterial activity, which proved that 伪 -helix was an antibacterial functional domain of HMG N2. The second part is the construction of HMG N2 伪 -helix domain and rich histone 5 chimeric antibacterial polypeptide. The fusion peptide prokaryotic expression plasmid was constructed by PCR splicing technique.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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