【作者】 李明；

【导师】 王伯瑶；

【作者基本信息】 四川大学 ， 病理学与病理生理学， 2005， 博士

【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更多还原

【Abstract】 Our environment is contaminated by anumerous mumber and variety of pathogenic microorganisms. In resent years, the increasing frequency of bacteria infections in patients together with the emergence of strains resistant to currently used antibiotics. It is an urgent need to discover a new class of antimicrobial drug.Most of endogenous antimicrobial peptides are small cationic peptides, widely distributed all over the body and play a major role in innate immunity. Antimicrobial peptides exhibit remarkable antibacterial, antifungal, antiparasite and antiviral activity against a variety of microorganisms and parasites. However, the antimicrobial activity and the chemical structure of individual antimicrobial peptide can be altered by ions, serum factors and proteases. So, development of a new kind of antibacterial polypeptides through combinations of two or more peptides may be a new way to find new drugs for treatment of microorganism, especially antibiotic-resistant strain infection.In addition to microbicidial activity, Defensins, one of an inportant antimicrobial peptide classes in humans, may also take part in adaptive immune response as a regulators and enhancers. Using p-defensin as immune adjuvant may develop a new DNA vaccine that enhances anti-cancer immune response.In this study, a novel chimeric antimicrobial polypeptide His5-a is constructed to enhance its antimicrobial activity and its chemical stability via the combination of Histatin 5, an antimicrobial peptide derived from human saliva, and HMG N2 α-helical domain that was recently identified to be an antimicrobial peptide from human cervical mucus in our laboratory. And, a chimeric DNAvaccine pcDNA 3.1/hBD2-PSMA is also dveloped to enhance anti-cancer immunity agaist prostate cancer through fusing PSMA, a prostate specific membrane antigen, and human P-denfensin-2.This study includes three parts.Part I is to perfome the N-terminal sequencing of a newly identified antimicrobial polypeptide in our laboratory from human cervical mucus and isolate its full length of cDNA for the use in construction of the chimeric antimicrobial polypeptide. The obtained N-terminal sequence of the newly isolated antibacterial peptide is PKRKADGEAK. Then, degenerate primer was designed and the full length of cDNA was amplified using 3’ RACE PCR method. This peptide was identified as High Mobility Group Chromosal protein N2 (HMG N2) by BLAST software analysis. OMIGA protein structure softwar analysis indicated a a-helix region, located from 17th to 47th amino acid reside, was contained in HMG N2. The antibacterial assay showed that only the a-helic structure (17 to 47) had antibacterial activity among three synthetic short peptides (1 to 16, 17 to 47, 48 to 90). The a-helical structure may be antibacterial domain ofHMGN2.Part His to construct chmeric protein prokaryotic expressing plasmid by combination of Histatin 5 and high mobility group chromosal protein N2 a helix through PCR SOEing method. E.coli BL21 strain carrying the recombinant plasmid was cultured in LB medium for 6 hours in the presence of IPTG to induce protein expression. The bacteria were lysed by freezing /thawing in the presence of lysozyme, The fusion protein was separated by affinity chromatography, and tag protein was cleaved by thrombin digestion, at last the recombinant His5-a chimeric peptide was purified using reverse-phase high performance liquid chromatography. In comparison with Histatin 5 alone, the chimeric polypeptide had a higher antibacterial and antifungal activity, and was more stable to tempratuer and trypsin than histatin 5. Using Scan electron microscope, we found that the chimeric peptide was not membrane active for its antimicrobial activity. DNA bingding assay showed that the chimeric peptide binded to bacterial DNA. Fleurecence labling and laser confacal microscopicobservation showed that the chimeric peptide distributed in the cytoplasm and nuclear structure of microorgansms. All these results suggested that targeting and attacking microbial DNA and organelle would be the antimicrobial mechanisms of the chimeric His5-a peptide.The aim of Part mis to construct DNA vaccine containing prostate-specific membrane antigen and hBD-2. PSMA expression could be detected in the DNA vaccine-ingected tissues in two weeks after the last immunization. PSMA specific antibody was detected in the serum of the immunized mice. T cells of the immunized mice were amplificated, especially the percentage of CD4+ T cells was markedly increased. Cytotoxity assay indicated that cytotoxic T lymphocytes to PSMA-expressing cells was also induced. This result provided evendence that P-defensin could be used as a new type of adjuvant in antitumor immunity.更多还原