舍蝇（Musca domestica vicina）幼虫抗菌肽的提取及其生物活性的初步研究
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摘要
本课题采用针刺损伤及金黄色葡萄球菌感染诱导方法，首次从舍蝇(Musca domestica vicina)幼虫提取到抗菌肽。这些抗菌肽是通过加热、阳离子交换柱层析及Sephadex G-50葡聚糖凝胶柱层析等步骤初步分离纯化得到，经电泳检测可看到2条电泳带，其一分子量为4kD，另一为9.6kD。舍蝇幼虫抗菌肽能有效地抑制金黄色葡萄球菌生长，但是对大肠杆菌、绿脓杆菌和枯草芽孢杆菌均无抑制作用。此外，它们还能抑制人胃癌细胞MGC80-3和BGC-823，人乳腺癌细胞MCF-7及人肺癌细胞SPC-A-1的体外增殖，并且呈浓度依赖关系。当舍蝇幼虫抗菌肽浓度低于或为62.5μg/mL时，细胞死亡率无明显变化；但是当浓度提高至125μg/mL时，细胞死亡率均有很大提高，分别为41.18％，28.62％，40.41％和37.61％；而且舍蝇幼虫抗菌肽浓度最大(250μg/mL)时，细胞死亡率都超过67％。
本课题还发现不经诱导的舍蝇幼虫也能产生抗菌肽，其中用麦麸饲养的舍蝇幼虫提取液的抑菌圈直径为8mm，而用猪粪饲养的抑菌圈直径为15mm，几乎是前者的2倍。
The preliminary studies on antimicrobial peptides
isolated from Musca domestict, vicina
Abstract
In this research, two antimicrobial peptides were detected in the extraction of the Musca domestica vicina larvae when the larvae were injured with a needle and then infected by Staph vlococcus auerus. These peptides were isolated by a three steps protocol including heating, CM- cellulose cation-exchange chromatography, and Sephadex G-50 gel filtration. The molecular weight of these peptides are 4kD and 9.6kD respectively. They can kill Staphylococcus auerus effectively, but no effect on Bacillus sub tills, Escherchia coil and Pseudornonas aeruginosa. The pept ides also can inhibit the proliferation of stomach cancer cells MGC8O-3 and BGC-823, breast cancer cells MCF-7 and lung cancer cells SPC-A-l in vitro. When the concentrations lower than 62.5ug/mL, their inhibition rates are under 23%, but when the concentrations rise to 125ug/mL, all of the
inhibition rates increase about 20%. When their
concentrations are as high as 250ug/mL, the inhibition rates all higher than 67%. We also found the larvae of Afusca domestica vicina without inducing also can produce the antimicrobial peptides. The diameter of inhibition zone of the extraction of the larvae feeding by bran is 8mm, but that of the larvae feeding by pig dejection is 15mm.
本课题还发现不经诱导的舍蝇幼虫也能产生抗菌肽，其中用麦麸饲养的舍蝇幼虫提取液的抑菌圈直径为8mm，而用猪粪饲养的抑菌圈直径为15mm，几乎是前者的2倍。
The preliminary studies on antimicrobial peptides
isolated from Musca domestict, vicina
Abstract
In this research, two antimicrobial peptides were detected in the extraction of the Musca domestica vicina larvae when the larvae were injured with a needle and then infected by Staph vlococcus auerus. These peptides were isolated by a three steps protocol including heating, CM- cellulose cation-exchange chromatography, and Sephadex G-50 gel filtration. The molecular weight of these peptides are 4kD and 9.6kD respectively. They can kill Staphylococcus auerus effectively, but no effect on Bacillus sub tills, Escherchia coil and Pseudornonas aeruginosa. The pept ides also can inhibit the proliferation of stomach cancer cells MGC8O-3 and BGC-823, breast cancer cells MCF-7 and lung cancer cells SPC-A-l in vitro. When the concentrations lower than 62.5ug/mL, their inhibition rates are under 23%, but when the concentrations rise to 125ug/mL, all of the
inhibition rates increase about 20%. When their
concentrations are as high as 250ug/mL, the inhibition rates all higher than 67%. We also found the larvae of Afusca domestica vicina without inducing also can produce the antimicrobial peptides. The diameter of inhibition zone of the extraction of the larvae feeding by bran is 8mm, but that of the larvae feeding by pig dejection is 15mm.
引文
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28． Ohshima M., Mitsuhara I, Okamoto M, et al. Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect. Journal of Biochemistry. 1999, 125(3) :431-435
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Sarcophaga peregrina (flesh fly) larvae. J Biol Chem . 1985,260(12) :7174-7177
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2． Baquero F. ,Blazquez J. Evolution of antibiotic resistance. Tree. 1997, 12 (12) : 482-487
3． Boman H. G. Peptide antibiotics and their role in innate immunity. Curr Biol. 1995,3:435-448
4． Bulet P.,Hetru C. ,Dimarcq J.L. , et al. Antimicrobial peptides in insect: structure and function.Developmen and Comparative Immunology. 1999,23:329-344
5． Bulet P.,Urge L.,Ohresser S.,et al. Enlarged scale chemical synthesis and range of activity of drosocin, an O-glycosylated antibacterial peptide of Drosophila. Eur J Biochem. 1996,238:64-69
6． Cociancich S.,Ghazi A.,Hetru C.,et al. Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. Journal of Biological Chemistry. 1993,268:19239-19245
7． Cole A.M.,Weis P.,Diamond G. , et al. Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions fo winter flouder. J Biol Chem. 1997,272:12008-12013
8． De Lucca A. J. , Bland J. M. , Jacks J. T. , et al. Fungicidal activity of cecropin A. Antimicrob Agents Chemother. 1997,41:481-483
9． Diaz-Achirica P.,Vach J.,Guinea A. The plasma membrane of leishmania donovani promastigotes hybrid peptide. Biochemical Journal. 1998, 330(1) :453-460
10． Dimarcq J. L. , Hoffmann D..Meister M. , et al. Characterization and transcriptional profile of a Drosophila gene encoding an insect defensin. A study in insect immunity. Eur J Biochem. 1994,221:201-209
11． Fehlbaum P. Bulet P.,Michaut L.,et al. Insect immunity:septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. Journal of Biological Chemistry. 1994,269:33159-33163
12． Ferrandon D. , Jung A.C. , Criqui M.,et al. A drosomycin-GFP reporter transgene reveals a local immmune response in Drosophila that is not dependent on the Toll pathway. EMEO Journal. 1998,17:1217-1227
13． Hara S. , Yamakawa M. Moricin, a novel type of antibacterial peptide isolated from the silkworm, Bombyx mori. J Biol Chem. 1995, 270(50) :29923-29927
14． Hultmark D. , Steiner H. , Rasmusson T.,et al. Insect immunity: purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. European Journal of Biochemistry. 1980,106:7-16
15． Jaynes J.M.,Nagapala P., Destefanobeltran L., et al. Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Science. 1993, 89(1) :43-53
16． Kanai A.,Natori S. Analysis of a gene cluster for Sarcotoxin II, a group of antibacterial proteins of Sarcophage peregrina. Mol Cell Biol. 1990, 10(12) :6114-6122
17． Kylsten P. , Smarkovlis C.,Hultmark D. The cecropin locus in Drosophila:a compact gene cluster involved in the response to infection.EMBO J. 1990,9:217-224
18． Lambert J. , Keppi E. ,Dimarcq J.L. et al.Insect immunity:isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bacterial peptides. Proc Nail Acad Sci USA.1989,86:262-266
19． Lamberty M. , Ades S.,Joseph S.U.,et al. Insect immunity:isolation from the leptdopteran Heltothis virescens of a novel insect defensin with potent antifungal activity. J Biol Chem 1999,274(140:9320-9326
20． Lee S. R. , Kurata S. , Natori S. Molecular cloning of Cdna FOR sapecin B, an antibacterial protein of Sarcophaga, and its detection in larval brain.FEBS Letter. 1995,368(3) :485-487
21． Lehane M.J. , Wu D. , Lehane S. M.Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans. Proc Natl Acad Sci USA. 1997,94:11502-11507
22． Lemaitre B., Reichhart J.M., Hoffman J. A. Drosophila host defense: Differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Natl Acad Sci USA. 1997,94:14614-14619
23． Levashina E. A. , Ohresser S.,Bulet P.,et al. Metchnikowin, a novel immune-inducible probline-rich peptide from Drosophila with antibacterial and antifungal properties. Eur J Biochem. 1995,233:694-700
24． Marchini D. , Giordano P. C.,Amons R. , et al. Purification and primary structure of ceratotoxin A and B,two antibacterial peptides from the female reproductive accessory glands of the medfly Ceratitis capitata (Insecta: Diptera).Insect Biochem Mol Biol. 1993, 23(5) : 591-598
25． Marchini D., Marri L. , Rosetto M. , et al. Presence of antibacterial peptides on the laid egg chorion of the medfly Cretitis capitata. Biochemical and Biophysical Research Communications. 1997,240:657-663
26． Matsuyama K.,Natori S. Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina. J Biol Chem. 1988,263(32) : 17117-17121
27． Mitsuhara I. , Nakajima Y.,Natori S.,et al. In vitro growth inhibition of human intestinal bacteria by Sarcotoxin IA, an insect bactericidal peptide. Biotechnology Letters. 2001,23(7) :569-573
28． Ohshima M., Mitsuhara I, Okamoto M, et al. Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect. Journal of Biochemistry. 1999, 125(3) :431-435
29． Okada M.,Natori S. Primary structure of sarcotoxin I, an antibacterial protein induved in the hemolymph of
Sarcophaga peregrina (flesh fly) larvae. J Biol Chem . 1985,260(12) :7174-7177
30． Okada M. , Natori S. Purification and characterization of an antibacterial protein from haemolymph of Sarcophaga peregrina (flesh-fly) larvae. Biochem J. 1983,211:727-734
31． Ourth D.D., Renis H. E. Antiviral melanization reaction of heliothis-virescens hemolymph against DNA and RNA viruses in-vitro. Comparative Biochemistry and Physiology. 1993, 105:719-723
32． Ourth D. D.,Renis H. E. Antiviral response of heliothis-virescens larvae. American zoologist. 1991, 31 (5) :A90-A90
33． Park C. B. , Kim H. S., Kim S. C., et al. Mechanism of action of the antimicrobial peptide buforin II :buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochemical and Biophysical Research Communications. 1998,244(1) :253-257
34． Possani L. D. ,Zurita M. , Delepierre M. , et al. From noxiustoxin to Shiva-3, a peptide toxic to the sporogonic development of Plasmodium berghei. Toxicon. 1998,36(11) : 1683-1692
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