【作者】 梁永利；

【导师】 王金星；

【作者基本信息】 山东大学 ， 动物学， 2005， 博士

【摘要】 自然界中大多数多细胞生物都生活在与病原微生物经常接触的环境中。这些生物的生存依赖于体内多种成分参与的宿主防御体系。高等脊椎动物的宿主防御依赖于两种类型的免疫反应:天然免疫和获得性免疫。而在昆虫等无脊椎动物中,宿主防御仅仅依赖于天然免疫。 天然免疫的一个主要机制为在生物体内组成性或诱导性表达一些内源肽,它们构成了机体防御病原体的快速而高效的手段,这些肽被称为抗菌肽。抗菌肽构成了原始的免疫反应机制,在从人到植物再到昆虫的真核生物中广泛存在,一些微生物中也出现了它们的踪迹。抗菌肽具有分子量小、等电点高、热稳定好、在生理条件下多数带正电荷、广谱抗菌等特点。它们不仅对革兰氏阳性菌和革兰氏阴性菌有较强的杀灭作用,对某些真菌、原生动物,尤其对耐药性细菌也具有杀灭作用。随着新的疾病不断产生,细菌的抗性已成为医学中不断增加的威胁。由于抗菌肽具有广谱的抗菌活性,被认为是解决病原微生物对抗生素不断增强的抗性问题的很好选择。一些抗菌肽还表现出了抗肿瘤、抗病毒和防治寄生虫传染病方面的应用潜力。在治疗HIV等性传播疾病方面也展现出了良好的应用前景。在农业生产中,它不仅可以用于动物饲料和食品保鲜,将抗菌肽基因转入动物和植物体中可以有效地防治一些病害的发生。人们对于抗菌肽的广泛研究还增加了人们对于先天性免疫系统的认知。因此,对抗菌肽的研究具有重要的理论意义和应用价值。 家蝇与人类的生活密切相关。家蝇从幼虫到成虫均生活在杂菌横生的环境里,是许多病原体的携带者,约100多个引起人类和动物疾病的病原体与家蝇有关,包括伤寒、霍乱、杆菌性痢疾、肺结核、炭疽热、婴儿腹泻和一些寄生虫病等。由于家蝇自身不受这些病原微生物的感染,人们推测家蝇体内具有独特的免疫防御机制,体内可能产生活性较强的抗菌肽。我们实验室曾从家蝇中纯化到1条分子量为10KDa的抗菌肽,它具有热稳定性,100℃加热10min仍有活性,表现出抗革兰氏阴性菌和革兰氏阳性菌的潜力。之后,我们实验室从家蝇中克隆出1条防御素基因(GenBank no.AY260152),命名为家蝇防御素Md defensin(Mdde),和1条天蚕素基因(GenBank no.AF416602),命名为家蝇天蚕素Md-Cecropin(Md-Cec)。其它学者还从家蝇中克隆出了家蝇攻击素基因(GenBank no.AY460106和GenBank no.DQ062744)。 本研究应用RT-PCR和原位杂交技术研究家蝇抗菌肽基因在家蝇体内的表达模式,对比经微生物诱导前后,以及诱导后不同时间家蝇体内抗菌肽基因表达量的变化,调查抗菌肽基因在家蝇体内的组织分布情况。并应用RT-PCR克更多还原

【Abstract】 The most of multicellular organisms live in surroundings laden with pathogens. The survival of them depends on host defense mechanisms involving various components in vivo. In high vertebrates such as mammalian, the host defense systems depend on two types of immune: the innate immunity and the acquired immunity. But the defenses of invertebrate, such as insects, only depend on innate immunity.A character of the innate immunity is that endogenous peptides are constitutively expressed or induced, which provide a fast and effective means of defence against pathogens. This group of peptides termed ’antimicrobial peptides’ (AMPs). AMPs make up of a primitive immune mechanism and are found in a wide range of eukaryotic organisms, from humans to plants and insects. Some of them are found in microorganisms. Most of these peptides have small molecular weight, high isoelectric point, heat stability, overall net positive charge under the physiological condition, a broad antimicrobial spectrum and so on. They not only function on Gram-positive bacteria and Gram-negative bacteria, but also on fungi, protozoa, especially bacteria with drug resistance. With the continual emergence of new diseases, bacterial resistance has become a growing threat to human health. Due to their broad-spectrum of antimicrobial activities, the AMPs were considered to be excellent candidates for potential novel antibiotic agents. Some AMPs also exhibit tumouricidal and virucidal properties, and have potential of prevention and cure of parasite infectious disease. A few of these peptides show activity against pathogens causing sexually transmitted infection, including HIV. In agricultural production, they not only are used as animal feed additive or antistaling agent for food storage, but also in transgenic animals or plants to prevent diseases. The extensive studies on AMPs have helped us to understand the innate immunity well.Therefore, the studies in AMPs have important academic value and good application prospect.The houseflies (Musca domestica) that live in surroundings full of various microbes are closely correlative with human life. Houseflies have been implicated in the spread of over 100 pathogens that may cause diseases in humans and animals, including typhoid, cholera, bacillary dysentery, tuberculosis, anthrax ophthalmia and infantile diarrhea, as well as parasitic worms. But they can thrive without causing infection. Therefore it is presumed that the houseflies have unique immune defense mechanism and can product antimicrobial peptides with strong activity. Our laboratory has purified an antimicrobial peptide, with a molecular weight of 10 kDa. The peptide was heat stable, and had activities that were retained after 10 min incubation at 100 °C. It showed potential activities against Gram-positive and Gram-negative bacteria. After that, our lab also cloned a defensin gene (GenBank no. AY260152), named Md defensin {Mdde), and a cecropin gene (GenBank no. AF416602), named Md-Cecropin (Md-Cec), from housefly. Otherwise, attacin genes (GenBank no. AY460106 and GenBank no. DQ062744) were cloned by other authors form housefly.In this study, the expression patterns of the genes encoding cecropin, defensin and attacin were studied by semi-quantitative RT-PCR and in situ hybridization. We analysed the effects of challenge with the mixtures of Escherichia coli and Staphylococcus aureus on the antimicrobial peptides mRNA transcription in the larvae of M. domestica, and investigated the tissue distributions of the gene transcripts in larvae of M. domestica. The cDNA sequence encoding mature attacin peptide was cloned by RT-PCR, and then expressed in prokaryotic expression system.The results of the studies are as follows.1. RT-PCR was performed using Md-Cec specific primers MdcecF/MdcecR and total RNA from the unchallenged and challenged larvae at 5h, lOh, 24h and 48h after challenge respectively. The results showed that the Md-cec gene expression was detectable in challenged larvae from 5h to 24h after infection challenge, whereas not detectable in native larvae and challenged larvae at 48h. Quantitativeanalysis by Labwork v4.5 revealed that the quantity of Md-Cec transcript increased rapidly in 5h after challenge, then decreased slowly, maintained to lOh, distinctly reduced at 24h, no detectable again at 48h.2. In order to localize expression of Md-Cec mRNA in different tissues, we performed in situ hybridization with Md-Cec antisense DIG RNA probes and the tissue slices of larvae unchallenged and challenged with the mixtures of E. coli and 5. aureus. The results showed that there was strong transcription in the fat body, and that transcript was also detected in the epithelia of the body wall and epidermis of gut. However, transcript was not observed in muscles, or trachea in the challenged larvae. In unchallenged flies, no transcript was detected in any tissue.3. To analyze the effects of challenge with the mixtures of E. coli and S1. aureus on the Mdde transcripts in the larvae of M. domestica, RT-PCR was performed using Mdde specific primers Mdde F/Mdde R and total RNA from the unchallenged and challenged larvae respectively at 5h, lOh, 24h and 48h after challenge. The results of RT-PCR showed that the Mdde transcripts were found in either the challenged larvae with the mixtures of E. coli and S. aureus from 5h to 48h or unchallenged insects, and that the transcript level of the gene rapidly increased at 5h after challenge, largely increased at 48h.4. To study the tissue distributions of the Mdde transcripts in larvae of housefly, the in situ hybridization with Mdde antisense DIG RNA probes were carried out. The results of the in situ hybridization showed that the gene of Mdde was transcribed mainly in the epithelia of the body wall and the fat body, and no transcription signal was detected in tracheae, gut and muscles. The transcripts of Mdde in the epithelia of the body wall were found both in challenged larvae and unchallenged larvae, whereas the transcripts of Mdde in fat body were found only in challenged larvae.5. RT-PCR was performed using housefly attacin specific primers Attacin F/Attacin R and total RNA from the whole body of the unchallenged larvae and the tissues of challenged larvae at lOh after challenge by E.coli. The results showed that the attacin gene expression was not detectable in native larvae, and was detectable in fat body of challenged larvae, whereas not detectable in body wall andgut of challenged larvae.6. A cDNA encoding mature attacin peptide of housefly (Musca domestica) was isolated from total RNA of challenged larvae by E. coli, using RT-PCR, and named Mdatt. The sequence alignment showed that the nucleotide sequence of Mdatt is 97% identical to M. domestica attacin (GenBank no. DQ062744) and 96% to another M. domestica attacin(Gen&ank no. AY460106), while the deduced amino acid sequence of Mdatt is 99% and 98% identical respectively to GenBank DQ062744 and GenBank AY460106. The Mdatt was cloned into the pGEX-4T-l vector and expressed in E.coli BL21. The result of SDS-PAGE showed that the expressed production resided in the host cells in the form of inclusion bodies.Base on the analyses of the above results, the conclusions we come up with are as follows.1. The transcription of the Md-cec is inducible by microbe infections. The rapidly transcription occur in 5h after challenge by bacteria. The quantities of the transcript maintain to lOh, distinctly reduce at 24h, and come back to native level at 48h.2. The distribution of the Md-Cec transcript has tissue specificity. The transcript mainly produces in fat body, body wall and midgut, not in muscles and trachea.3. The transcription of Mdde in larvae of M domestica is constitutive. But the transcript quantity is inducible to increase by microorganism. The quantity gradually increases after challenge by bacteria, rapidly increases in 5h, largely increases at 48h.4. The Mdde transcript also has tissue specificity, mainly in body wall and fat body. The transcription is low level constitutive expression in body wall, whereas inducible in fat body. Differing from Md-cec, the transcript of Mdde is not found in gut. We consider that the transcription pattern of the Mdde in larvae of M. domestica is the results of the insect adapting to the natural environment.5. The transcription of the attacin is inducible by microbe infections in larvae of M. domestica. The transcript mainly produces in fat body, not in body wall and gut.6. The cDNA sequence encoding mature attacin peptide is conserved, and the amino acid sequence more conserved. The mature attacin peptide can be expressed in prokaryotic expression system.This study will enhance the understanding of the immune system in housefly;enrich the knowledge of the innate immunity in insects. The recombinant expressed antimicrobial peptides can be used in continued studies, and large-scale production may be used in genetic engineered medicine and animal feed additives.更多还原