【作者】 孙龙；

【导师】 冯颖；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2012， 博士

【摘要】 抗菌肽是由基因编码的一类小分子多肽，在昆虫先天性免疫系统中占有重要地位，通常由10-50个氨基酸组成，多数为碱性、阳离子肽，带2-7个正电荷，具有水溶性好、热稳定性高、活性广泛、细菌不易产生耐药性等优点，极有可能成为新一代抗生素的替代药物，因而成为生命科学、药学和免疫学研究的热点之一。昆虫是自然界种类最多、数量最大的生物类群，同时也是抗菌肽未被充分挖掘的宝库。从丰富的昆虫资源中分离、提取和鉴定具有生物活性的抗菌肽或先导化合物是昆虫抗菌肽药物研究和开发的重要步骤。本论文以鞘翅目昆虫黄粉虫（Tenebrio molitor Linnaeus）幼虫、喙尾琵甲（Blapsrhynchoptera Fairmaire）幼虫为研究材料，通过金黄色葡萄球菌（Staphylococcus aureus)和大肠杆菌(Escherichia coli)混合液针刺诱导使其产生抗菌肽，采用不同溶液对其体内的抗菌肽进行提取，经多次凝胶色谱、反相色谱、高效液相色谱，成功地从黄粉虫幼虫、喙尾琵甲幼虫粗提物中获得了具有明显抑制革兰氏阳性菌的小分子肽类，主要研究结果如下：（1）抗菌肽最佳提取溶液的筛选：通过实验比较了10%乙酸溶液、0.1%三氟乙酸溶液、Tris-HCl（pH8.0）、Tris-HCl（pH6.5）等4种不同的溶液对黄粉虫幼虫抗菌肽的提取效果，结果发现10%乙酸粗提物和10%乙酸空白对照都有抑菌作用，导致对抑菌活性的归属产生严重干扰，因而不宜做提取溶液；其余3种提取液只有0.1%三氟乙酸提取物经后续凝胶柱预纯化后，能够检测到明显的抑菌活性，所以确定0.1%三氟乙酸作为2种昆虫抗菌肽的最佳提取溶液。（2）抗菌肽的预纯化及Tricine-SDS-PAGE：黄粉虫幼虫0.1%三氟乙酸粗提物经SephadexG50凝胶层析后，非诱导组和诱导组均得到2个峰，其中第二个峰TFADZP2、TFAYDP2对金黄色葡萄球菌、枯草芽孢杆菌表现出明显的抑制作用，对大肠杆菌、绿脓杆菌无活性；喙尾琵甲幼虫0.1%三氟乙酸粗提物经Superose12凝胶柱预纯化后，非诱导组和诱导组均得到3个峰，其中第一个峰HWDZP1、HWYDP1对金黄色葡萄球菌、枯草芽孢杆菌表现出抑制作用，对大肠杆菌无活性，此外诱导组HWYDP1还对绿脓杆菌表现出抑制作用；Tricine-SDS-PAGE结果表明，黄粉虫幼虫抗菌肽TFADZP2、TFAYDP2主要由分子量40kD以下的小肽组成，喙尾琵甲抗菌肽HWDZP1、HWYDP1主要由分子量20kD以下的小肽组成。（3）抗菌肽诱导组与非诱导组活性的比较：2种昆虫诱导组和非诱导组粗提物经纯化后都能检测到抑菌活性，而且诱导组在较低浓度时对革兰氏阳性菌的抑菌圈直径明显大于非诱导组，抑菌活性较强，表明实验得到的2种昆虫抗菌肽在体内都有基础表达，经菌液诱导后活性有所增强。对喙尾琵甲来说，诱导组还增加了对绿脓杆菌的抑制活性，说明诱导可能导致抑菌谱扩大。（4）黄粉虫抗菌肽的纯化及鉴定：经两步Superdex Peptide凝胶色谱、一步反相色谱和一步高效液相色谱，从黄粉虫幼虫诱导组纯化出一个具革兰氏阳性菌抑制活性的组分，命名为TFAYDP2-2-1-1-2，液相色谱检测纯度为单一组分，质谱显示其分子量为2552.5Da，属于小分子抗菌肽，该组分对金黄色葡萄球菌、枯草芽孢杆菌的最小抑制浓度分别为10μg/mL和20μg/mL，对革兰氏阴性菌大肠杆菌、绿脓杆菌无活性。目前鞘翅目昆虫还未见如此低分子量的抗菌肽报道。（5）喙尾琵甲抗菌肽的纯化：喙尾琵甲幼虫诱导组预纯化组分经一步反相色谱纯化，得到组分HWYDP1-1，对金黄色葡萄球菌具有明显的抑制作用。综上所述，采用0.1%三氟乙酸作为黄粉虫幼虫抗菌肽的提取溶液，通过两步凝胶柱层析、一步反相色谱和一步高效液相色谱从黄粉虫幼虫抗菌肽粗提物中获得了一个纯化组分，具有较强的抑制金黄色葡萄球菌和枯草芽胞杆菌活性，对这两种革兰氏阳性菌的最小抑制浓度分别是10μg/mL和20μg/mL。但是该组分对革兰氏阴性菌大肠杆菌、绿脓杆菌无活性；质谱测定其分子量为2552.5Da，属于小分子量抗菌肽，目前鞘翅目昆虫体内未发现如此低分子量的抗菌肽。采用0.1%三氟乙酸溶液对喙尾琵甲幼虫抗菌肽进行了提取，通过两步凝胶柱层析、一步反相色谱从喙尾琵甲幼虫抗菌肽粗提物中获得一具有抑制金黄色葡萄球菌的组分。更多还原

【Abstract】 Antimicrobial peptides (AMPs) defined as small peptides of10to50amino acid residues,have played a particularly important role in innate immune system of insects. Generallyspeaking, most of AMPs are encoded by single genes that comprise highly homologous genefamilies. They are usually basic and cationic, with a net positive charge of+2to+7owing to anexcess of basic amino acids (artinine, lysine and histidine) over acidic amino acids. Besidestheir high antimicrobial activities against Gram-negative and/or Gram-positive bacteria, thesepeptides also have a diverse range of targets, including fungi, viruses, parasites such astrypanosomes and plasmodia, and even cancer cell. As the difference on the mechanisms ofaction against bacteria from conventional antibiotics, which targeted mainly cellular membrane,AMPs can be expected to develop as promising candidates against antibiotic-resistantpathogens that are often not sensitive to classical antibiotic treatment. Therefore the researchon AMPs is becoming one of the hottest fields in the world. Insects are not only the biggestgroup within the animal kingdom, but also a treasure of various antimicrobial peptides whichhas not been fully excavated. For decades, one major area of interest for the discovery andstudy of new antibiotics was the investigation of AMPs derived from abundant insectresources.In this disserdation, extraction, purification and identification of AMPs from the larvae oftwo Coleopteran insects, Tenebrio molitor Linnaeus (T. molitor) and Blaps rhynchopteraFairmaire (B. rhynchoptera), were systematically studied. Firstly we challenged the larvae withthe combined suspension of living Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus) by pricking their abdomens with a stainless needle. Then extraction was carried outexactly24hours after the induction Four kinds of solutions were used to extract AMPs from T.molitor larvae. After repeated gel chromatography, reversed phase chromatography, higeperformance liquid chromatography (HPLC), a partly characterized small peptide was obtained from crude extracts of T. molitor, with strong anti-Gram-positive bacteria activities. Maincontents and results are listed as follows:(1) Screening of the optimum extraction solution of AMPs from T. molitorFour solutions,10%acetic acid,0.1%trifluoroacetic acid, Tris-HCl (pH8.0), andTris-HCl (pH6.5), were employed in extraction process of AMPs from T. molitor in order toselect the suitable extraction solution. The results indicated that10%acetic acid itself showedantimicrobial activity, leading to difficulty in judging inhibitory effect, and should not beselected as the extraction solution. Only0.1%trifluoroacetic acid extract among the rest threesolutions was found to display detectable inhibitory activity against bacteria afterpre-purification. Therefore,0.1%trifluoroacetic acid was the optimum extraction solution for T.molitor AMPs.Considering that both T. molitor and B. rhynchoptera belong to same order,0.1%trifluoroacetic acid was directly applied as extracted solution for B. rhynchoptera AMPs.(2) Pre-purification of two crude AMPs extracts by gel chromatography andTricine-SDS-PAGE analysisCrude extracts of T. molitor AMPs, coming from both induced and non-induced groupswere separated into two fractions after SephadexG50gel chromatography, and the second peak,marked as TFADZP2and TFAYDP2, respectively, showed obviously inhibitory activitiesagainst S. aureus and B. subtilis, but no anti-E. coli and anti-P. aeruginosa activities.Similarly, crude extracts of B. rhynchoptera AMPs from induced group and non-induced groupwere separated into three fractions after Superose12gel chromatography. The first peak,marked as HWDZP1and HWYDP1, respectively, had strong anti-S. aureus and anti-B. subtilisactivities, but no inhibitory effect against E. coli. In addition, HWYDP1from the inducedgroup displayed anti-P. aeruginosa activity. Tricine-SDS-PAGE analysis suggested thatTFADZP2and TFAYDP2mainly composed of small peptides with molecular mass below40kD, HWDZP1HWYDP1mainly consisted of small peptides with molecular mass less than20kD.(3) Comparison of antibacterial activity between induced group and non-induced group The antibacterial activities were detectable in induced group as well as the non-inducedfor these two kinds of insects. More interestingly, the fraction from the induced group hadbigger zone of inhibition against bacteria than the one from non-induced group at a lowerconcentration. The result implied that the AMPs encoded by gene in T. molitor and B.rhynchoptera were constitutively expressed and their activities could be enhanced greatly afterinduction with microbes. Additionally, the fraction from induced group of B. rhynchopteraexhibited anti-P. aeruginosa except anti-Gram-positive bacteria, suggesting a broader spectrumprobably occurred after challenge with miroorganisms.(4) Purification and Characterization of T. molitor AMPsBy two-step of Superdex Peptide, one-step of reversed-phase chromatography and highperformance liquid chromatography, a purified fraction with strong anti-Gram-positive bacteriaactivity was obtained from the induced group of T. molitor, named TFAYDP2-2-1-1-2. Thisfraction was partially characterized. The analysis of matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) indicated that its molecular mass was2552.5Da, a small molecule antimicrobial peptides. The fraction had no inhibitory effect onGram-negative bacteria E. coli and P. aeruginosa, but had apparent activity against S. aureusand B. subtilis and the minimal inhibitory concentrations were10μg/mL and20μg/mL,respectively. To the best of our knowledge, no antimicrobial peptide with ultra-low molecularmass has reported so far in the whole Coleopteran insects.(5) Purification of B. rhynchoptera AMPsBy one-step of reversed-phase chromatography, a component, named HWYDP1-1, wasgained from the induced group of B. rhynchoptera and displayed significantly inhibitoryactivity against S. aureus.In short, a homogeneous AMP from T. molitor larvae was achieved after the followingprocedures: extracted with acidic solution (0.1%trifluoroacetic acid), pre-purified by two stepsof gel chromatography, purified by one step of reversed-phase chromatography and one step ofhigh performance liquid chromatography. This AMP was proved to have strong inhibitory effects on S. aureus and B. subtilis, and the minimal inhibitory concentrations were10μg/mLand20μg/mL, respectively, but had no activity against E.coli and P aeruginosa. Its molecularmass was determined as2552.5Da by MALDI-TOF MS. It is probably a novel AMP fromColeoptera. Similar extraction and purification programs was carried out on AMPs of B.rhynchoptera. A fraction was gained with inhibitory activity against S. aureus.更多还原