家蝇（Musca domestica）抗菌蛋白的分离纯化及部分性质研究
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摘要
本文首次从家蝇(Musca domestica)幼虫血淋巴中分离纯化到一种具抗菌活性的蛋白质，并对其部分理化和生物学性质进行了研究。通过体壁损伤法诱导家蝇幼虫产生免疫血淋巴，经沸水浴热变性、减压蒸馏浓缩、CM-Sepharose离子交换层析、Sephadex G-50凝胶过滤等步骤纯化后，聚丙烯酰胺凝胶电泳证明为单一蛋白区带。
经SDS-PAGE测得该抗菌蛋白的分子量为12,600道尔顿，IEF测得其等电点为9.8。氨基酸组成分析表明，家蝇抗菌蛋白富含脯氨酸，其含量达27.3％。NR／R双向SDS-PAGE证明家蝇抗菌蛋白分子中的两个半胱氨酸残基未形成二硫键。远紫外圆二色谱(CD)分析显示，在生理条件下，家蝇抗菌蛋白的二级结构构象组成为：26.6％α-螺旋，23.7％β-折叠，49.7％β-转角与无规卷曲。在接近细胞膜的疏水环境中，β-折叠含量减少，α-螺旋含量增加。推测家蝇抗菌蛋白的抗菌机理是可能作用于细菌细胞膜，α-螺旋在这一过程中起着重要的作用。
对数种细菌的抗菌活性检测表明，家蝇抗菌蛋白具有较广的抗菌谱，对人病原细菌、昆虫病原细菌及非病原细菌都有抗性，对革兰氏阳性菌的抗性高于革兰氏阴性菌。它不具血细胞凝集活性，亦不能使血细胞发生溶血，具有很高的热稳定性。这种蛋白质不是溶菌酶，而是一种未见报道的抗菌蛋白，根据其各项性质认为它可能属于富含脯氨酸的抗菌蛋白类。
昆虫抗菌蛋白具有传统抗生素不可比拟的优点，有望成为新一代抗菌药物。我们的工作为家蝇免疫机制的进一步深入研究打下了基础，对家蝇抗菌蛋白的潜在应用价值的开发有着重要意义。
The immunized haemolymph was produced from Musca domestica(House Fly) larvae by inducement of injuring the body wall with a hypodermic needle. A protein with antibacterial activity was purified at the first time and the properties of the protein had been studied. The protein was obtained by means of boiled water bath, CM-Sepharose ion-exchange chromatography and Sephadex G-50 gel filtration. The purified protein moved as a single band in low pH PAGE. Its molecular weight was 12,600 Dalton by SDS-PAGE and its isoelectric point was 9.8 by IEF. Amino acid composition assay showed that it was rich in proline. NR/R 2-dimensional SDS-PAGE proved that the two cysteine residues in the molecule were not engaged in intramolecular disulfide bridge. The far UV CD analysis indicated that the protein contained 26.6%
α-helix, 23.7% β-sheet, 49.7% β-turn and random coil at pH7. In the hydrophobic environments similar to the membrane of bacteria cell, the content of a-helix increased and the content of P
-sheet decreased. It was inferred that the helix should be important to the antibacterial activity. The protein had broad antibacterial activity against several human pathogens, insect pathogens and non-pathogens, and the activity against gram-positive bacteria was higher than the activity against gram-negative bacteria. It was not a lectin, nor a lysozyme, but an unknown antibacterial protein with high heat-stability, which should belong to the pro-rich antibacterial protein family.
经SDS-PAGE测得该抗菌蛋白的分子量为12,600道尔顿，IEF测得其等电点为9.8。氨基酸组成分析表明，家蝇抗菌蛋白富含脯氨酸，其含量达27.3％。NR／R双向SDS-PAGE证明家蝇抗菌蛋白分子中的两个半胱氨酸残基未形成二硫键。远紫外圆二色谱(CD)分析显示，在生理条件下，家蝇抗菌蛋白的二级结构构象组成为：26.6％α-螺旋，23.7％β-折叠，49.7％β-转角与无规卷曲。在接近细胞膜的疏水环境中，β-折叠含量减少，α-螺旋含量增加。推测家蝇抗菌蛋白的抗菌机理是可能作用于细菌细胞膜，α-螺旋在这一过程中起着重要的作用。
对数种细菌的抗菌活性检测表明，家蝇抗菌蛋白具有较广的抗菌谱，对人病原细菌、昆虫病原细菌及非病原细菌都有抗性，对革兰氏阳性菌的抗性高于革兰氏阴性菌。它不具血细胞凝集活性，亦不能使血细胞发生溶血，具有很高的热稳定性。这种蛋白质不是溶菌酶，而是一种未见报道的抗菌蛋白，根据其各项性质认为它可能属于富含脯氨酸的抗菌蛋白类。
昆虫抗菌蛋白具有传统抗生素不可比拟的优点，有望成为新一代抗菌药物。我们的工作为家蝇免疫机制的进一步深入研究打下了基础，对家蝇抗菌蛋白的潜在应用价值的开发有着重要意义。
The immunized haemolymph was produced from Musca domestica(House Fly) larvae by inducement of injuring the body wall with a hypodermic needle. A protein with antibacterial activity was purified at the first time and the properties of the protein had been studied. The protein was obtained by means of boiled water bath, CM-Sepharose ion-exchange chromatography and Sephadex G-50 gel filtration. The purified protein moved as a single band in low pH PAGE. Its molecular weight was 12,600 Dalton by SDS-PAGE and its isoelectric point was 9.8 by IEF. Amino acid composition assay showed that it was rich in proline. NR/R 2-dimensional SDS-PAGE proved that the two cysteine residues in the molecule were not engaged in intramolecular disulfide bridge. The far UV CD analysis indicated that the protein contained 26.6%
α-helix, 23.7% β-sheet, 49.7% β-turn and random coil at pH7. In the hydrophobic environments similar to the membrane of bacteria cell, the content of a-helix increased and the content of P
-sheet decreased. It was inferred that the helix should be important to the antibacterial activity. The protein had broad antibacterial activity against several human pathogens, insect pathogens and non-pathogens, and the activity against gram-positive bacteria was higher than the activity against gram-negative bacteria. It was not a lectin, nor a lysozyme, but an unknown antibacterial protein with high heat-stability, which should belong to the pro-rich antibacterial protein family.
引文
[1]Hultmart D, Boman H G, et al. Purification and properties three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 1980, 106:7-16
[2]黄自然，陈劲伟．昆虫防御机制研究的一些进展．生物学通报，1990，(10)：9-10
[3]Yamakawa M. Insect antibacterial proteins: Regulatory mechanisms of their synthesis and a possibility as new antibiotics. J Seric Sci Jpn. 1998,67(3): 163-182
[4]Boman H G, Hultmart D. Cell-free immunity in insects. Ann Rev Microbiol. 1987,41:103-126
[5]王远程，刘伟，杨峰等．家蝇血淋巴的提取及抗菌物质的诱导．微生物学报，1992，32(6)：439-444
[6]周永富，饶军华，李文楚等．家蝇抗菌物质的诱导．生物学杂志，1997，14(3)：23-26
[7]王远程，左晓峰，孙东旭等．家蝇幼虫抗菌物质组成及其理化性质．微生物学报，1997，37(2)：148-153
[8]饶军华，周永富，郑青．家蝇免疫血淋巴的性质研究．昆虫天敌，1999，21(3)：121-125
[9]赵东红，戴祝英，周开亚．昆虫抗菌肽的功能、作用机理与分子生物学研究最新进展．生物工程进展，1999，19(5)：14-17
[10]李阜棣，喻子牛，何绍江主编．农业微生物学实验技术．北京：中国农业出版社，1996，305-308
[11]黄自然，郑庭辉，梁怡章等．柞蚕抗菌肽的抑菌效应．科学通报．1986，14：1107-1109
[12]Laemmli U K. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature. 1970,227:680-683
[13]Cleveland D W, Fischer S G, Laemmli U K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem, 1977,252(3): 1102-1106
[14]夏其昌主编．蛋白质化学研究技术与进展．北京：科学出版社，1999，102～104
[15]何忠效，张树政主编．电泳，北京：科学出版社，1990，36～39
[16]Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem. 1976,72:248-254
[17]陈曾燮等编．生物化学实验．合肥：中国科学技术大学出版社，1994，32-36
[18]王希成，王帆，邹晓明等．氧化型肌酸激酶的研究．中国科学，1993，23：1279-1286
[19]Cheng Y H, Yang J T, Chau K H. Determination of the helix and β form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974,13(6):3350-3359
[20]Steiner H. Secondary structure of the Cecropins: antibacterial peptides from Hyalophora cecropia. FEBS Lett. 1982,137:246-248
[21]施庆洛，屈贤铭，鲁子贤．中国柞蚕杀菌肽D的构象分析．生物化学与生物物理学报．1986，18(2)：210-212
[22]Moore S, Stein W H. Chromatographic determination of amino acids by use of automatic recording equipment. Methods Enzyme. 1963,6:819-848
[23]孙册等编．凝集素．北京：科学出版社，1986，20-21
[24]张纪忠主编．微生物分类学．上海：复旦大学出版社1990，16-50
[25]Boman H G, Faye I, Gudmundsson G H. Cell-free immunity in Cecropia.: A model system for antibacterial proteins. Eur J Biochem. 1991,201:23-31
[26]Hultmark D. Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 1993,9(5): 178-183
[27]Hoffmann J A, Hetru C, Reichhart J M. The humoral antibacterial response of Drosophila. FEBS Lett. 1993,325(1-2):63-66
[28]Ip Y T, Levine M. Molecular genetics of Drosophila immunity. Curt Opin Genet Dev. 1994,4(5):672-677
[29]Hoffmann J A, Reichhart J M, Hetru C. Innate immunity in higher insects. Curt Opin Immunol. 1996,8(1):8-13
[30]Hoffmann D, Hultmart D, Boman H G. Insect immunity: Galleria mellonella and other lepidoptera have cecropia-P9-like factors active against gram negative bacteria. Insect Biochem. 1981,11:537-538
[31]Flyg C, Dalhammar G, Boman H G. Insect immunity: Inducible antibacterial activity in Drosophila. Insect Biochem. 1987,17:153-160
[32]Chadwick J M, Aston W P. An overview of insect immunity. Animal Models of Comparative and Developmental Aspects of Immunity and Disease, ed. M E Gershwin, E L Cooper. 1979, New York: Pergamon. 1-14
[33]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
[34]Qu X-M, Steiner H, Engstrom A. Insect immunity: isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth, Antheraea pernyi. Eur J Biochem. 1982,127:219-224
[35]Dickinson L, Russell V, Durra P E. A family of bacteria-regulated, cecropin D-like peptides from Manduca sexta. J Biol Chem. 1988,21:19424-19429
[36]Boman H G. Antibacterial peptides: key components needed in immunity. Cell.1991, 65(2):205-207
[37]Hultmart D, Engstrom A. Isolation and structure of Cecropin D and four miner antibacterial components from cecropia pupae. Eur J Biochem. 1982,127:207-217
[38]赵小凡，王金星，王绪英等．柞蚕抗菌蛋白纯化及性质．山东大学学报．1999，34(3)：339-343
[39]Powning R F, Davidson W J. Studies in insect bacteriolytic enzyme-Ⅱ. Some
physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella. Comp Biochem Physiol. 1976,B 55:221-228
[40]Fennell J F, Shipman W H, Cole L J. Antibacterial action of melittin, a polypeptide from bee venom. Proc Soc Exp Biol Med. 1968,127(3):707-710
[41]屈贤铭，唐海伦Steiner H．柞蚕杀菌肽B、D对脂质体的作用．生物化学与生物物理学报．1989，21：35-42
[42]Hoffmann J A, Hetru C. Insect defensins: inducible antibacterial peptides. Immunol Today. 1992,13(10):411-415
[43]Cornet B, Bonmatin J M, Hertru C. Refined three-dimensional structure of insect defensin A in water from NMR data. Struture. 1995,3(5):435-448
[44]张双全，屈贤铭．昆虫免疫应答及抗菌肽应用前景．生物化学杂志，1987，3(1)：11-18
[45]鲁子贤，崔涛，施庆洛编著．圆二色性和旋光色散在分子生物学中的应用．北京：科学出版社，1987：50-73
[46]Okada M, Natori S. Mode of action of a bactericidal protein induced in the haemolymph of Sarcophaga peregrina(flesh-fly)larvae. Biochem J. 1984,222:119-124
[47]Christensen B, Fink J, Merrifield RB. Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc Natl Acad Sci U S A. 1988,85(14):5072-5076
[48]Fink J, Merrifield R B, Boman A. The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity. J Biol Chem. 1989 264(11):6260-6267
[49]Cociancich S, Ghazi A, Hetru C. Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus.J Biol Chem. 1993, 268(26): 19239-19245
[50]Matsuyama K, Natori S. Mode of action of sapecin, a novel antibacterial protein of Sarcophaga peregrina (flesh fly). J Biochem (Tokyo). 1990,108(1):128-132
[51]Casteels P, Ampe C, Jacobs F. Apidaecins: antibacterial peptides from honeybees. EMBO J. 1989,8(8):2387-2391
[52]Casteels P, Ampe C, Riviere L. Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera). Eur J Biochem. 1990,187(2):381-386
[53]Bulet P, Dimarcq J L, Hetru C. A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution. J Biol Chem. 1993,268(20): 14893-14897
[54]Levashina E A, Ohresser S, Bulet P. Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties. Eur J Biochem. 1995,233(2):694-700
[55]Cociancich S, Dupont A, Hegy G. Novel inducible antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus. Biochem J. 1994,300 (Pt 2):567-575
[56]Hara S, Yamakawa M. A novel antibacterial peptide family isolated from the silkworm, Bombyx mori. Biochem J. 1995,310 (Pt 2):651-656
[2]黄自然，陈劲伟．昆虫防御机制研究的一些进展．生物学通报，1990，(10)：9-10
[3]Yamakawa M. Insect antibacterial proteins: Regulatory mechanisms of their synthesis and a possibility as new antibiotics. J Seric Sci Jpn. 1998,67(3): 163-182
[4]Boman H G, Hultmart D. Cell-free immunity in insects. Ann Rev Microbiol. 1987,41:103-126
[5]王远程，刘伟，杨峰等．家蝇血淋巴的提取及抗菌物质的诱导．微生物学报，1992，32(6)：439-444
[6]周永富，饶军华，李文楚等．家蝇抗菌物质的诱导．生物学杂志，1997，14(3)：23-26
[7]王远程，左晓峰，孙东旭等．家蝇幼虫抗菌物质组成及其理化性质．微生物学报，1997，37(2)：148-153
[8]饶军华，周永富，郑青．家蝇免疫血淋巴的性质研究．昆虫天敌，1999，21(3)：121-125
[9]赵东红，戴祝英，周开亚．昆虫抗菌肽的功能、作用机理与分子生物学研究最新进展．生物工程进展，1999，19(5)：14-17
[10]李阜棣，喻子牛，何绍江主编．农业微生物学实验技术．北京：中国农业出版社，1996，305-308
[11]黄自然，郑庭辉，梁怡章等．柞蚕抗菌肽的抑菌效应．科学通报．1986，14：1107-1109
[12]Laemmli U K. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature. 1970,227:680-683
[13]Cleveland D W, Fischer S G, Laemmli U K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem, 1977,252(3): 1102-1106
[14]夏其昌主编．蛋白质化学研究技术与进展．北京：科学出版社，1999，102～104
[15]何忠效，张树政主编．电泳，北京：科学出版社，1990，36～39
[16]Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem. 1976,72:248-254
[17]陈曾燮等编．生物化学实验．合肥：中国科学技术大学出版社，1994，32-36
[18]王希成，王帆，邹晓明等．氧化型肌酸激酶的研究．中国科学，1993，23：1279-1286
[19]Cheng Y H, Yang J T, Chau K H. Determination of the helix and β form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974,13(6):3350-3359
[20]Steiner H. Secondary structure of the Cecropins: antibacterial peptides from Hyalophora cecropia. FEBS Lett. 1982,137:246-248
[21]施庆洛，屈贤铭，鲁子贤．中国柞蚕杀菌肽D的构象分析．生物化学与生物物理学报．1986，18(2)：210-212
[22]Moore S, Stein W H. Chromatographic determination of amino acids by use of automatic recording equipment. Methods Enzyme. 1963,6:819-848
[23]孙册等编．凝集素．北京：科学出版社，1986，20-21
[24]张纪忠主编．微生物分类学．上海：复旦大学出版社1990，16-50
[25]Boman H G, Faye I, Gudmundsson G H. Cell-free immunity in Cecropia.: A model system for antibacterial proteins. Eur J Biochem. 1991,201:23-31
[26]Hultmark D. Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 1993,9(5): 178-183
[27]Hoffmann J A, Hetru C, Reichhart J M. The humoral antibacterial response of Drosophila. FEBS Lett. 1993,325(1-2):63-66
[28]Ip Y T, Levine M. Molecular genetics of Drosophila immunity. Curt Opin Genet Dev. 1994,4(5):672-677
[29]Hoffmann J A, Reichhart J M, Hetru C. Innate immunity in higher insects. Curt Opin Immunol. 1996,8(1):8-13
[30]Hoffmann D, Hultmart D, Boman H G. Insect immunity: Galleria mellonella and other lepidoptera have cecropia-P9-like factors active against gram negative bacteria. Insect Biochem. 1981,11:537-538
[31]Flyg C, Dalhammar G, Boman H G. Insect immunity: Inducible antibacterial activity in Drosophila. Insect Biochem. 1987,17:153-160
[32]Chadwick J M, Aston W P. An overview of insect immunity. Animal Models of Comparative and Developmental Aspects of Immunity and Disease, ed. M E Gershwin, E L Cooper. 1979, New York: Pergamon. 1-14
[33]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
[34]Qu X-M, Steiner H, Engstrom A. Insect immunity: isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth, Antheraea pernyi. Eur J Biochem. 1982,127:219-224
[35]Dickinson L, Russell V, Durra P E. A family of bacteria-regulated, cecropin D-like peptides from Manduca sexta. J Biol Chem. 1988,21:19424-19429
[36]Boman H G. Antibacterial peptides: key components needed in immunity. Cell.1991, 65(2):205-207
[37]Hultmart D, Engstrom A. Isolation and structure of Cecropin D and four miner antibacterial components from cecropia pupae. Eur J Biochem. 1982,127:207-217
[38]赵小凡，王金星，王绪英等．柞蚕抗菌蛋白纯化及性质．山东大学学报．1999，34(3)：339-343
[39]Powning R F, Davidson W J. Studies in insect bacteriolytic enzyme-Ⅱ. Some
physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella. Comp Biochem Physiol. 1976,B 55:221-228
[40]Fennell J F, Shipman W H, Cole L J. Antibacterial action of melittin, a polypeptide from bee venom. Proc Soc Exp Biol Med. 1968,127(3):707-710
[41]屈贤铭，唐海伦Steiner H．柞蚕杀菌肽B、D对脂质体的作用．生物化学与生物物理学报．1989，21：35-42
[42]Hoffmann J A, Hetru C. Insect defensins: inducible antibacterial peptides. Immunol Today. 1992,13(10):411-415
[43]Cornet B, Bonmatin J M, Hertru C. Refined three-dimensional structure of insect defensin A in water from NMR data. Struture. 1995,3(5):435-448
[44]张双全，屈贤铭．昆虫免疫应答及抗菌肽应用前景．生物化学杂志，1987，3(1)：11-18
[45]鲁子贤，崔涛，施庆洛编著．圆二色性和旋光色散在分子生物学中的应用．北京：科学出版社，1987：50-73
[46]Okada M, Natori S. Mode of action of a bactericidal protein induced in the haemolymph of Sarcophaga peregrina(flesh-fly)larvae. Biochem J. 1984,222:119-124
[47]Christensen B, Fink J, Merrifield RB. Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc Natl Acad Sci U S A. 1988,85(14):5072-5076
[48]Fink J, Merrifield R B, Boman A. The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity. J Biol Chem. 1989 264(11):6260-6267
[49]Cociancich S, Ghazi A, Hetru C. Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus.J Biol Chem. 1993, 268(26): 19239-19245
[50]Matsuyama K, Natori S. Mode of action of sapecin, a novel antibacterial protein of Sarcophaga peregrina (flesh fly). J Biochem (Tokyo). 1990,108(1):128-132
[51]Casteels P, Ampe C, Jacobs F. Apidaecins: antibacterial peptides from honeybees. EMBO J. 1989,8(8):2387-2391
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