大孔吸附树脂吸附分离家蝇抗菌肽的研究
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摘要
昆虫抗菌肽是昆虫免疫防御系统的重要成分,具有相对分子质量小、热稳定性好、抗菌谱广等特点。家蝇存在独特的免疫机制,在我国分布广,资源丰富,容易养殖、成本低,是一个等待开发的良好资源。因此对家蝇抗菌肽分离纯化的研究具有重要理论意义和实际应用价值。
家蝇抗菌肽的抑菌活性与其疏水性有一定的联系,故采用大孔吸附树脂根据疏水性的差异来从家蝇粗蛋白中分离家蝇抗菌肽,并对大孔吸附树脂的分离工艺进行了优化选择,然后通过RP-HPLC进一步纯化,得到了抑菌活性最高的组分。
本论文考察多种大孔吸附树脂对家蝇蛋白的吸附性能,D101大孔吸附树脂对家蝇蛋白的吸附量可达217.18 mg/g,乙醇是比较理想的洗脱剂,随着乙醇的浓度增加,洗脱率也相应提高,当用75%乙醇洗脱时,洗脱率为75.70%。通过试验确定D101大孔吸附树脂富集分离家蝇抗菌肽的上样条件为:上样流速0.5 BV/h(BV, Bed Volume,床体积),上样浓度为15 mg/mL。D101大孔吸附树脂富集分离洗脱采用15%、35%和55%的乙醇分级洗脱,经过这三种浓度的乙醇溶液洗脱后得到的组分的蛋白质含量分别为39.40%、71.72%和72.74%,而粗家蝇蛋白的蛋白质含量只有12.79%,说明洗脱后的蛋白质的含量有很大的提高,同时其疏水性值也从粗家蝇蛋白的4.17 kJ/mol分别增加到了4.30 kJ/mol、4.43 kJ/mol和4.75 kJ/mol。随着疏水性的提高,家蝇抗菌肽的抑菌活性也呈现增加的趋势,其中55%乙醇洗脱组分的抑菌活性最高。
经过分离纯化使用后的树脂再吸附性能降低,用常规的再生方法不能恢复大孔吸附树脂的吸附能力,研究分别采用酸醇法和碱醇法对D101大孔吸附树脂再生条件进行优化:酸醇法对D101大孔吸附树脂的静态再生条件为HCl浓度0.1 mol/L,乙醇浓度85%,温度30℃,浸泡时间6 h,再生液用量50 mL/g干树脂;动态再生条件为再生液流速1 BV/h,再生液用量66.7 mL/g,其他条件同静态再生。碱醇法对D101大孔吸附树脂的静态再生条件为NaOH浓度0.1 mol/L,乙醇浓度85%,温度为30℃,浸泡时间6 h,再生液用量66.7 mL/g干树脂;动态再生条件为再生液流速为1 BV/h,其他条件同静态再生。在上述条件下,同时使用酸醇法和碱醇法对D101大孔吸附树脂进行再生,使用20次后树脂的吸附性能仅降低1.5%。
对D101大孔吸附树脂富集分离的55%乙醇洗脱组分进一步进行RP-HPLC纯化,得到的多个峰都具有抑菌活性,其中F12的抑菌活性最高。经过二次RP-HPLC和SDS-PAGE电泳检测,该峰达到色谱纯和电泳纯,测得其相对分子质量为4842。氨基酸分析表明,F12富含碱性氨基酸和疏水性氨基酸,疏水性值为5.82 kJ/mol。与D101大孔吸附树脂分离组分相比,F12的疏水性增加更为明显,其抑菌活性也有大幅度的提高。测定各种组分对各试验菌的MIC(Minimal Inhibitory Concentration,最低抑菌浓度)可以知F12对大肠杆菌的MIC最低,为30μg/mL。同时,试验证明F12具有良好的耐高温、耐低温和耐酸碱性,F12在pH 9左右抑菌活性最大。
Insect antibacterial peptides are the important components of insect immune systems, and have advantages of small molecule weight, heat-stability and broad antibacterial activity. Musca domestica has the strong disease- resistance and the unique immunity mechanism,distributes widely in China, is easy to culture, and low-cost, thus the housefly is a good resource deserving to be explored. Study on the antibacterial peptide from the housefly is both theoretically and practically important.
Antibacterial Peptides from Musca domestica larvae was separated and purified by macroporous adsorption resin chromatography, of which antibacterial activity has particular relationship with its hydrophobic property. The optimized parameters for the processing of the active compounds in antibacterial peptides separated by maroporous adsorption resin chromatography were known by test. The more active peptide obtained was purified by RP-HPLC further, and the most antibacterial peptides was acquired.
Adsorbed ability of macroporous adsorption resin chromatography was recognized. The adsorbing quantity of D101 macroporous adsorption resin chromatography for the Antibacterial Peptides reached 217.18 mg/g. Ethanol was a variety of optimized eluted solution. Increased with concentration of ethanol, the eluted efficiency was elevated. Eluted efficiency of Antibacterial Peptides with D101 macroporous adsorption resin was 75.70%, when eluted by 75% concentration ethanol. The best loading parameters for peptides on D101 macroporous adsorption resin was 15mg/mL sample concentration with 0.5 BV/h (BV, Bed Volume) loading rate. The 15%、35% and 55% ethanol solution was used to elute on D101 macroporous adsorption resin. The protein content of compounds separated by D101 macroporous adsorption resin with 15%、35% and 55% ethanol solution were 39.40%, 71.72% and 72.74% respectively, while the protein content of the origin Antibacterial Peptides was 12.79%, indicated that the protein content was increased after eluting. At the same time, hydrophobic value of compounds eluted by the three different concentration ethanol was 4.30、4.43 and 4.75 kJ/mol, 0.13~0.64 kJ/mol increased than the origin Antibacterial Peptides, which suggested that the antibacterial activity of Antibacterial Peptides was higher with hydrophobic value increased. The compounds eluted by 55% ethanol owned the best antibacterial activity.
The adsorbed capacity decreased dramaticly after separation and purify, but the traditional methods would not recover the adsorbed capacity of D101 macroporous adsorption resin. The optimized condition for reuse method with acid and cedrol was investigated as following: with the static reuse, the concentration of HCL was 0.1mol/L, the concentration and quantity of ethanol was 85% and 50 mL/g dry resin at 30℃for six hours; with dynamic reuse, the rate of reuse solution was 1 BV/h and the quantity of ethanol was 66.7 mL/g dry resin, and the other parameters was same as the static reuse .The optimized condition for reuse method with base and cedrol was investigated as following: with the static reuse, the concentration of NaOH was 0.1 mol/L, the concentration and quantity of ethanol was 85% and 66.7 mL/g dry resin at 30℃for six hours; with dynamic reuse, rate of reuse solution was 1 BV/h and the other parameters was same as the static reuse. Both reuse method with acid and cedrol and with base and cedrol used to reuse D101 macroporous adsorption resin, the adsorbed capacity reduced by 1.5% after employing 20 times.
RP-HPLC was applied to purify the compound obtained by D101 macroporous adsorption resin with 55% ethanol, the peak of F12 was the highest antibacterial activity among the peaks with Antibacterial activity. After the second RP-HPLC and SDS-PAGE electrophoresis test, chromatographically and electrophoretically pure ingredient was detected with molar weight of 4842. Analysis of amino acid showed that F12 was rich in basic amino acid and hydrophobic amino acid and hydrophobic value was 5.82 kJ/mol, indicated that it was a basic antibacterial peptide with more hydrophoby. Compared with the compound separated by D101 macroporous adsorption resin, the hydrophobicity and antibacterial activity of F12 was enhanced. The MIC for test organism of F12 was the lowest among each ingredients, which was 30μg/mL. The ingredient of F12 was insensitive to either high or zero temperature and acid or basic envirment, with the most antibacterial activity at pH 9.
家蝇抗菌肽的抑菌活性与其疏水性有一定的联系,故采用大孔吸附树脂根据疏水性的差异来从家蝇粗蛋白中分离家蝇抗菌肽,并对大孔吸附树脂的分离工艺进行了优化选择,然后通过RP-HPLC进一步纯化,得到了抑菌活性最高的组分。
本论文考察多种大孔吸附树脂对家蝇蛋白的吸附性能,D101大孔吸附树脂对家蝇蛋白的吸附量可达217.18 mg/g,乙醇是比较理想的洗脱剂,随着乙醇的浓度增加,洗脱率也相应提高,当用75%乙醇洗脱时,洗脱率为75.70%。通过试验确定D101大孔吸附树脂富集分离家蝇抗菌肽的上样条件为:上样流速0.5 BV/h(BV, Bed Volume,床体积),上样浓度为15 mg/mL。D101大孔吸附树脂富集分离洗脱采用15%、35%和55%的乙醇分级洗脱,经过这三种浓度的乙醇溶液洗脱后得到的组分的蛋白质含量分别为39.40%、71.72%和72.74%,而粗家蝇蛋白的蛋白质含量只有12.79%,说明洗脱后的蛋白质的含量有很大的提高,同时其疏水性值也从粗家蝇蛋白的4.17 kJ/mol分别增加到了4.30 kJ/mol、4.43 kJ/mol和4.75 kJ/mol。随着疏水性的提高,家蝇抗菌肽的抑菌活性也呈现增加的趋势,其中55%乙醇洗脱组分的抑菌活性最高。
经过分离纯化使用后的树脂再吸附性能降低,用常规的再生方法不能恢复大孔吸附树脂的吸附能力,研究分别采用酸醇法和碱醇法对D101大孔吸附树脂再生条件进行优化:酸醇法对D101大孔吸附树脂的静态再生条件为HCl浓度0.1 mol/L,乙醇浓度85%,温度30℃,浸泡时间6 h,再生液用量50 mL/g干树脂;动态再生条件为再生液流速1 BV/h,再生液用量66.7 mL/g,其他条件同静态再生。碱醇法对D101大孔吸附树脂的静态再生条件为NaOH浓度0.1 mol/L,乙醇浓度85%,温度为30℃,浸泡时间6 h,再生液用量66.7 mL/g干树脂;动态再生条件为再生液流速为1 BV/h,其他条件同静态再生。在上述条件下,同时使用酸醇法和碱醇法对D101大孔吸附树脂进行再生,使用20次后树脂的吸附性能仅降低1.5%。
对D101大孔吸附树脂富集分离的55%乙醇洗脱组分进一步进行RP-HPLC纯化,得到的多个峰都具有抑菌活性,其中F12的抑菌活性最高。经过二次RP-HPLC和SDS-PAGE电泳检测,该峰达到色谱纯和电泳纯,测得其相对分子质量为4842。氨基酸分析表明,F12富含碱性氨基酸和疏水性氨基酸,疏水性值为5.82 kJ/mol。与D101大孔吸附树脂分离组分相比,F12的疏水性增加更为明显,其抑菌活性也有大幅度的提高。测定各种组分对各试验菌的MIC(Minimal Inhibitory Concentration,最低抑菌浓度)可以知F12对大肠杆菌的MIC最低,为30μg/mL。同时,试验证明F12具有良好的耐高温、耐低温和耐酸碱性,F12在pH 9左右抑菌活性最大。
Insect antibacterial peptides are the important components of insect immune systems, and have advantages of small molecule weight, heat-stability and broad antibacterial activity. Musca domestica has the strong disease- resistance and the unique immunity mechanism,distributes widely in China, is easy to culture, and low-cost, thus the housefly is a good resource deserving to be explored. Study on the antibacterial peptide from the housefly is both theoretically and practically important.
Antibacterial Peptides from Musca domestica larvae was separated and purified by macroporous adsorption resin chromatography, of which antibacterial activity has particular relationship with its hydrophobic property. The optimized parameters for the processing of the active compounds in antibacterial peptides separated by maroporous adsorption resin chromatography were known by test. The more active peptide obtained was purified by RP-HPLC further, and the most antibacterial peptides was acquired.
Adsorbed ability of macroporous adsorption resin chromatography was recognized. The adsorbing quantity of D101 macroporous adsorption resin chromatography for the Antibacterial Peptides reached 217.18 mg/g. Ethanol was a variety of optimized eluted solution. Increased with concentration of ethanol, the eluted efficiency was elevated. Eluted efficiency of Antibacterial Peptides with D101 macroporous adsorption resin was 75.70%, when eluted by 75% concentration ethanol. The best loading parameters for peptides on D101 macroporous adsorption resin was 15mg/mL sample concentration with 0.5 BV/h (BV, Bed Volume) loading rate. The 15%、35% and 55% ethanol solution was used to elute on D101 macroporous adsorption resin. The protein content of compounds separated by D101 macroporous adsorption resin with 15%、35% and 55% ethanol solution were 39.40%, 71.72% and 72.74% respectively, while the protein content of the origin Antibacterial Peptides was 12.79%, indicated that the protein content was increased after eluting. At the same time, hydrophobic value of compounds eluted by the three different concentration ethanol was 4.30、4.43 and 4.75 kJ/mol, 0.13~0.64 kJ/mol increased than the origin Antibacterial Peptides, which suggested that the antibacterial activity of Antibacterial Peptides was higher with hydrophobic value increased. The compounds eluted by 55% ethanol owned the best antibacterial activity.
The adsorbed capacity decreased dramaticly after separation and purify, but the traditional methods would not recover the adsorbed capacity of D101 macroporous adsorption resin. The optimized condition for reuse method with acid and cedrol was investigated as following: with the static reuse, the concentration of HCL was 0.1mol/L, the concentration and quantity of ethanol was 85% and 50 mL/g dry resin at 30℃for six hours; with dynamic reuse, the rate of reuse solution was 1 BV/h and the quantity of ethanol was 66.7 mL/g dry resin, and the other parameters was same as the static reuse .The optimized condition for reuse method with base and cedrol was investigated as following: with the static reuse, the concentration of NaOH was 0.1 mol/L, the concentration and quantity of ethanol was 85% and 66.7 mL/g dry resin at 30℃for six hours; with dynamic reuse, rate of reuse solution was 1 BV/h and the other parameters was same as the static reuse. Both reuse method with acid and cedrol and with base and cedrol used to reuse D101 macroporous adsorption resin, the adsorbed capacity reduced by 1.5% after employing 20 times.
RP-HPLC was applied to purify the compound obtained by D101 macroporous adsorption resin with 55% ethanol, the peak of F12 was the highest antibacterial activity among the peaks with Antibacterial activity. After the second RP-HPLC and SDS-PAGE electrophoresis test, chromatographically and electrophoretically pure ingredient was detected with molar weight of 4842. Analysis of amino acid showed that F12 was rich in basic amino acid and hydrophobic amino acid and hydrophobic value was 5.82 kJ/mol, indicated that it was a basic antibacterial peptide with more hydrophoby. Compared with the compound separated by D101 macroporous adsorption resin, the hydrophobicity and antibacterial activity of F12 was enhanced. The MIC for test organism of F12 was the lowest among each ingredients, which was 30μg/mL. The ingredient of F12 was insensitive to either high or zero temperature and acid or basic envirment, with the most antibacterial activity at pH 9.
引文
1 Brahmachary M, Krishnan S P, Koh J L Y, et al. ANTIMIC: a database of antimicrobial sequences [J].Nucleic Acids Res, 2004, 32(1):586-589
2 Prates M V, Sforca M L, Regis W C. The NM R-derived solution structure of a new cationic antimicrobial peptide from the skin secretion of the anuran Hyla Punctata [J]. J Biol Chem, 2004, 279(13):13018
3 Lamberty M, Ades S, Uttenweiler-Joseph S, et al. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity [J]. J Biol Chem, 1999, 274 (14): 9320-9326.
4 Boman H G, Steiner H. Inducible antibacterial defense system in Drosophial[J]. Nature, 1972, 237: 232-235.
5 Osmanagaoglu O. Detection and characterization of Leucocin OZ, a new anti-listerial bacteriocin produced by Leuconostoc carnosum with a broad spectrum of activity [J]. Food Control, 2007, 18(2): 118-123
6 Dupuy B, Matamouros S. Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase [sigma]-factors [J]. Research in Microbiology, 2006. 157(3):201-205.
7 Bonn D. New bacteriocin from Greek cheese [J]. The Lancet Infectious Diseases, 2003. 3(2): 61-61.
8 Netz DJA, et al. Biochemical Characterisation and Genetic Analysis of Aureocin A53, a New, Atypical Bacteriocin from Staphylococcus aureus [J]. Journal of Molecular Biology, 2002. 319(3): 745-756.
9 Oscariz JC, Lasa I, Pisabarro AG. Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity [J]. FEMS Microbiology Letters, 1999, 178(2): 337-341.
10 Gomez S, Cosson C, Deschamps AM. Evidence for a bacteriocin-like substance produced by a newstrain of Streptococcus sp., inhibitory to Gram-positive food-borne pathogens [J]. Research in Microbiology, 1997, 148(9):757-766.
11 Ryan MP, Ross RP, Rea MC. New bacteriocin: (TEAGASC, Agriculture & Food Development Authority, Dublin 4, Ireland) PCT International Patent Application WO 96/32482 AI [J]. Trends in Food Science & Technology, 1997, 8(7): 248-248.
12 Huot E, Bacteriocin J. a New Bacteriocin Produced by Lactococcus lactisSubsp.cremorisJ46: Isolation and Characterization of the Protein and Its Gene [J]. Anaerobe, 1996, 2(3): p. 137-145.
13 Ali MF, Knoop F C, Vaudry H, et al. Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex[J]. Peptides, 2003, 24(7): 955-961.
14 Park S, Ahn HC, Kim S, et al. Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata [J]. FEBS Letters, 2001, 507(1): 95-100.
15 Suzuki S. Isolation and Characterization of Novel Antimicrobial Peptides, Rugosins A, B, and C, from the Skin of the Frog, Rana rugosa [J]. Biochemical and Biophysical Research Communications, 1995, 212(1): 249-254.
16 Zhou J, McClean S, Thompson A, et al., Purification and characterization of novel antimicrobial peptides from the skin secretion of Hylarana guentheri [J]. Peptides, In Press, Corrected Proof.
17 Zhou M, Chen T, Walker B, et al. Lividins: Novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida: Identification by "shotgun" cDNA cloning and sequence analysis [J]. Peptides, 2006, 27(9): 2118-2123.
18 Zhou M, Chen T, Walker B, et al. Pelophylaxins: Novel antimicrobial peptide homologs from the skin secretion of the Fukien gold-striped pond frog, Pelophylax plancyi fukienensis: Identification by "shotgun" cDNA cloning and sequence analysis [J]. Peptides, 2006, 27(1): 36-41.
19 Ahn H S, Cho W, Kang S H, et al., Design and synthesis of novel antimicrobial peptides on the basis of [alpha] helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study[J]. Peptides, 2006, 27(4): 640-648.
20 Fogaca A.C, Almeida I C, Eberlin M N, et al. Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases[J]. Peptides, 2006, 27(4): 667-674.
21 Inagaki H, Akagi M, Imai H, et al. Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia[J]. Archives of Biochemistry and Biophysics, 2004, 428(2): 170-178.
22 KonnoK, Hisada M, Fontana R, et al. Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis [J]. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001, 1550(1): 70-80.
23 Konno K, Hisada M, Naoki H, et al. Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus [J]. Peptides. In Press, Corrected Proof.
24 Park CB, Kim MS, Kim SC. A Novel Antimicrobial Peptide from Bufo gargarizans [J].Biochemical and Biophysical Research Communications, 1996, 218(1): 408-413.
25 Asiegbu F.O, Choi W, Li G, Nahalkova J, et al. Isolation of a novel antimicrobial peptide gene (Sp-AMP) homologue from Pinus sylvestris (Scots pine) following infection with the root rot fungus Heterobasidion annosum [J]. FEMS Microbiology Letters, 2003, 228(1): 27-31.
26 Iijima R, Kisugi J, Yamazaki M. A novel antimicrobial peptide from the sea hare Dolabella auricularia [J]. Developmental & Comparative Immunology, 2003, 27(4): 305-311.
27 Mahoney MM. Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide [J]. FEBS Letters, 1995, 377(3): 519-522.
28 Buhimschi IA, Jabr M, Buhimschi C S, et al. The novel antimicrobial peptide [beta]3-defensin is produced by the amnion: A possible role of the fetal membranes in innate immunity of the amniotic cavity [J]. American Journal of Obstetrics and Gynecology, 2004, 191(5): 1678-1687.
29 Cole A M, Kim Y H, Tahk S, Hong T, et al. Calcitermin, a novel antimicrobial peptide isolated from human airway secretions [J]. FEBS Letters, 2001, 504(1-2): 5-10.
30 Wehkamp J, Harder J, Meissner B, et al. Human [beta]-defessin 3: A novel antimicrobial peptide preferentially expressed in ulcerative celitis [J]. Gastroenterology, 2001, 120(5, Supplement 1): A182-A182.
31 Masschalck B, van Houdt R, Michiels C W. High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin [J]. Int J Food Microbiolm, 2001, 64(3):325
32 Zhengrong Hao, Irene Kasumba, Serap Aksoy. Proventriculus(cardia) plays a crucial role in immunity in tsetse[J]. Insect Biochemistry and Molecular Biology, 2003, 33:11455-1164
33 Zasloff, M. Antimicrobial peptides of multicellular organisms[J]. Nature, 2002, 415:389-395
34 Jacopo Vizioli, Adam M, Richman, et al. The defensin peptide of the malaria vector mosquito Anopheles gambiae :antimicrobial activities and expression in adult mosquitoes [J]. Insect Biochemistry and Molecular Biology, 2001, 31:241-248
35 Tanaka K. P13-kinase p85 is a target molecular of praline-rich antimicrobial peptide to suppress proliferation of ras-transformed cells[J]. Jpn,J, Cancer Res. 2001, 92:959-967
36 Mare Cudie, Barry A, Condie et al. Development of novel antibacterial peptides that kill resistant isolates[J]. Pepides, 2002, 23:2071-2083
37 Lockey T D. Formation of pores in Escherichia coli cell membranes by a cecropinisolated from hemolymph of Heliothis virescens larvae[J]. Journal of biochemistry, 236(1): 263-271.
38 Juvvadi P, Antibacterial peptides cecropin A-melittin hybrid analogues, C-terminal amides [J]. Journal of Peptide Science, 1996, 2(4): 223-232.
39 Shai Y, Rapaport D, Gazit E, et al. Molecular mechanism of membrane permeation by cytolytic toxins [J]. Toxicon, 1995, 33(3): p. 272-272
40程家安,唐振华.昆虫分子科学[M].科学出版社, 2001. 77-87
41董占鹏.蚕类抗菌肽及其研究进展[J].蚕学通讯. 2003, 23(3): 14-21
42 Christensen B, Fink J, Merrifield R.B. Channel-forming properties of cecropins and related compounds incorporated into compounds into planar lipid membranes [J]. Proc Natl Acad Sci, USA, 1988, 85: 5072-5076.
43 Clague M J, Cherry R J. A comparative study of band 3 aggregation in erythrocyte membranes by melittin and other cationic agents [J]. Biochimica Biophysica Acta, 1989, 980: 93-99.
44周义文,尹一兵,涂植光等.家蝇抗菌肽抗菌活性及抗菌机制的初步研究[J].中国抗生素杂志, 2004, 29(5): 272-274
45 Hou L X, Shi Y H, Zhai P, et al. Inhibition of foodborne pathogens by Hf-1, a novel antibacterial peptide from the larvae of the housefly (Musca domestica) in medium and orange juice[J]. Food control, 2007, 18(11):1350-1357
46盛长忠,安春菊,耿华等.一种家蝇幼虫热稳定抗菌肽的分离纯化[J].南开大学学报(自然科学版), 2002, 35(4): 6-10
47邓小娟,黄自然.柞蚕抗菌肽的分离纯化及杀菌效应[J].华南农业大学学报(自然科学版), 2002, 23(3):13-16
48宫霞,施用晖,乐国伟.家蝇幼虫抗菌肽MDL-1的分离纯化及其对大肠杆菌超微结构的影响[J].昆虫学报, 2004, 47(1): 8-13
49陆婕,汪俊汉,钟雅等.弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究[J].微生物学报, 2006, 46(3): 406-411
50张虹,柳正良,王洪泉.大孔吸附树脂在药学领域的应用[J].中国医药工业杂志, 2001, 32(1):4l-44.
51李伯庭,王湘,李小进.大孔吸附树脂在天然产物分离中的应用[J].中草药, 1990, 21(8):42-44.
52刘琦英.大孔吸附树脂提取酸性蛋白酶抑制剂研究[J].离子交换与吸附, 1989, 5(4): 251-255
53何炳林,英文强.离子交换与吸附树脂[M].上海科技教育出版杜,1995
54 Silva E M, Pompeub D R, Larondelle Y, et al. Optimisation of the adsorption of polyphenols from Inga edulis leaves on macroporous resins using an experimental design methodology [J]. Separation purification technology, 2007, 43: 274-280
55 Wan J B, Zhang Q W, Ye W C, et al. Quantification and separation of protopanaxatriol and protopanaxadiol type saponins from Panax notoginseng with macroporous resins[J]. Separation purification technology, 2007, in press
56 Gao M, Huang W, Liu C Z. Separation of scutellarin from crude extracts of Erigeronbreviscapus (vant.) Hand. Mazz. by macroporous resins[J]. Journal of Chromatography B, 2007, 858: 22-26
57 Fu B Q, Liu J, Li H, et al. The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid[J]. Journal of Chromatography A, 2005, 1089: 18-24
58钱宝庭.离子交换树脂应用技术[M].天津科学技术出版杜, 1984. 302-354
59解芳,周任重,袁勤生.大孔吸附树脂纯化SOD [J].中国医药工业杂志, 1995, 26(I): 1-2
60刘琦英.大孔吸附树脂提取酸性蛋白酶抑制剂研究[J].离子交换与吸附, 1989, 5(4): 251-255.
61李从军,鲁明波,余龙江等.大孔吸附树脂纯化谷胱甘肽的初步研究[J].食品科技, 2006, 10:70-73
62赵利,王璋,许时婴.大孔吸附树脂对酪蛋白非磷肽吸附性的研究[J].食品工业科技, 2002, 12: 48-31
63 Andres E, Dimarcq J L. Letters to the Editor--Clinical development of antimicrobial peptides [J]. International Journal of Antimicrobial Agents, 2005, 25: 448–452
64 Hansen L T, Austin J W, Gill T A. Antibacterial effect of protamine in combination with EDTA and refrigeration [J]. International Journal of Food Microbiology, 2001, 66(3): 149-161.
65 Cintas L M, Casaus P, Fernandez M F, et al. Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria [J]. Food Microbiology, 1998, 15(3): 289-298.
66徐飞,施文,王启松等.柞蚕抗菌肽D基因的合成[J].科学通报, 1988, 21: 15-17.
67赵东红,戴祝英,周开亚等.中国家蚕抗菌肽人工合成类CMⅣ基因在甜菜夜蛾虫体中的表达[J].微生物学报, 2001, 41(06): 680-685
68 Prates M V, Sforca M L, Regis W C. The N MR-derived solution structure of an newcationic antimicrobial peptide from the skin secretion of the anuran Hyla Punctata[J]. J Biol Chem, 2004, 279(13):1308-1302
69 Sherman R A, Hall M J, T homas S. Medicinal maggots: an anciert remedy for some contemporary afflictions [J]. Annu Rev Entomol, \ 2000, 45: 55– 81
70 Sukontason K, Vogtsberger R C, Sukontason K L, et al. Surface ultrastructure of the third-instar larvae of Hydrotaea spinigera Stein (Diptera:Muscidae), a fly species of forensic importance [J]. J Vector Ecol, 2001, 26 (2):191-195.
71刘健敏,钟芳,麻建国.大豆生理活性肽的研究(Ⅰ)—酶法水解的工艺[J].无锡轻工大学学报, 2004, 23(3): 41-45.
72赵亚华,等.生物化学实验技术教程[M].广州:华南理工大学出版社, 2000.
73 Walker J M. Protein determination by UV absorption[J]. From: The protocols handbook. Humana Press Inc,Totowa, NJ: 3-5.
74牟德海, OPA柱前衍生反相高效液相色谱法测定氨基酸含量[J].色谱, 1997, 15(4): 319-321.
75 Jens A N. Enzymic Hydrolysis of food proteins [M]. Elsevier Applied Science Publishers, 1986, 6.
76 Ney K H. Voraussage der bitterkeit von peptiden aus deren Aminosaure zusammensetzung[J]. Z.Lebensm Untersuch Forsch, 1971, 147: 64-71.
77 Hoffmann D, Hultmark D, Boman H G. Insect immunity: Galleria mellonella and other lep idop tea have Cecrop in-P9-like factors active against gram-negative bacteria [J]. Insect Biochem, 1981, 11:5370-548
78 Owen R. Fennema,王璋等译.食品化学[M].北京:中国轻工业出版社, 2003
79曾宪明.无机盐对大孔吸附树脂吸附人参总皂苷的影响[J].中国医药工业杂志, 1992, 23(8): 339-342.
80郭立安.高效液相色谱法纯化蛋白质理论与技术[M].陕西科学技术出版社, 1993.
81 Boman H G, Hultmark D, Steriner H. Insect immunity. Purification and properties of three inducible bactericial proteins from hemolymph of immunized pupae of Hyalophora cecropia [J]. European Journal of Biochemistry, 1980, 106: 7-16.
82金冬雁,黎孟枫译, .J.萨姆布鲁克, E.F弗里寺, T.曼尼阿蒂斯著.分子克隆[M].第二版,北京:科学出版社, 1992, 9: 08-913
83张卓然.主编临床微生物学和微生物学检验[M].第三版,北京:人民卫生出版社, 2003, 492-500
2 Prates M V, Sforca M L, Regis W C. The NM R-derived solution structure of a new cationic antimicrobial peptide from the skin secretion of the anuran Hyla Punctata [J]. J Biol Chem, 2004, 279(13):13018
3 Lamberty M, Ades S, Uttenweiler-Joseph S, et al. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity [J]. J Biol Chem, 1999, 274 (14): 9320-9326.
4 Boman H G, Steiner H. Inducible antibacterial defense system in Drosophial[J]. Nature, 1972, 237: 232-235.
5 Osmanagaoglu O. Detection and characterization of Leucocin OZ, a new anti-listerial bacteriocin produced by Leuconostoc carnosum with a broad spectrum of activity [J]. Food Control, 2007, 18(2): 118-123
6 Dupuy B, Matamouros S. Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase [sigma]-factors [J]. Research in Microbiology, 2006. 157(3):201-205.
7 Bonn D. New bacteriocin from Greek cheese [J]. The Lancet Infectious Diseases, 2003. 3(2): 61-61.
8 Netz DJA, et al. Biochemical Characterisation and Genetic Analysis of Aureocin A53, a New, Atypical Bacteriocin from Staphylococcus aureus [J]. Journal of Molecular Biology, 2002. 319(3): 745-756.
9 Oscariz JC, Lasa I, Pisabarro AG. Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity [J]. FEMS Microbiology Letters, 1999, 178(2): 337-341.
10 Gomez S, Cosson C, Deschamps AM. Evidence for a bacteriocin-like substance produced by a newstrain of Streptococcus sp., inhibitory to Gram-positive food-borne pathogens [J]. Research in Microbiology, 1997, 148(9):757-766.
11 Ryan MP, Ross RP, Rea MC. New bacteriocin: (TEAGASC, Agriculture & Food Development Authority, Dublin 4, Ireland) PCT International Patent Application WO 96/32482 AI [J]. Trends in Food Science & Technology, 1997, 8(7): 248-248.
12 Huot E, Bacteriocin J. a New Bacteriocin Produced by Lactococcus lactisSubsp.cremorisJ46: Isolation and Characterization of the Protein and Its Gene [J]. Anaerobe, 1996, 2(3): p. 137-145.
13 Ali MF, Knoop F C, Vaudry H, et al. Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex[J]. Peptides, 2003, 24(7): 955-961.
14 Park S, Ahn HC, Kim S, et al. Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata [J]. FEBS Letters, 2001, 507(1): 95-100.
15 Suzuki S. Isolation and Characterization of Novel Antimicrobial Peptides, Rugosins A, B, and C, from the Skin of the Frog, Rana rugosa [J]. Biochemical and Biophysical Research Communications, 1995, 212(1): 249-254.
16 Zhou J, McClean S, Thompson A, et al., Purification and characterization of novel antimicrobial peptides from the skin secretion of Hylarana guentheri [J]. Peptides, In Press, Corrected Proof.
17 Zhou M, Chen T, Walker B, et al. Lividins: Novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida: Identification by "shotgun" cDNA cloning and sequence analysis [J]. Peptides, 2006, 27(9): 2118-2123.
18 Zhou M, Chen T, Walker B, et al. Pelophylaxins: Novel antimicrobial peptide homologs from the skin secretion of the Fukien gold-striped pond frog, Pelophylax plancyi fukienensis: Identification by "shotgun" cDNA cloning and sequence analysis [J]. Peptides, 2006, 27(1): 36-41.
19 Ahn H S, Cho W, Kang S H, et al., Design and synthesis of novel antimicrobial peptides on the basis of [alpha] helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study[J]. Peptides, 2006, 27(4): 640-648.
20 Fogaca A.C, Almeida I C, Eberlin M N, et al. Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases[J]. Peptides, 2006, 27(4): 667-674.
21 Inagaki H, Akagi M, Imai H, et al. Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia[J]. Archives of Biochemistry and Biophysics, 2004, 428(2): 170-178.
22 KonnoK, Hisada M, Fontana R, et al. Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis [J]. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001, 1550(1): 70-80.
23 Konno K, Hisada M, Naoki H, et al. Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus [J]. Peptides. In Press, Corrected Proof.
24 Park CB, Kim MS, Kim SC. A Novel Antimicrobial Peptide from Bufo gargarizans [J].Biochemical and Biophysical Research Communications, 1996, 218(1): 408-413.
25 Asiegbu F.O, Choi W, Li G, Nahalkova J, et al. Isolation of a novel antimicrobial peptide gene (Sp-AMP) homologue from Pinus sylvestris (Scots pine) following infection with the root rot fungus Heterobasidion annosum [J]. FEMS Microbiology Letters, 2003, 228(1): 27-31.
26 Iijima R, Kisugi J, Yamazaki M. A novel antimicrobial peptide from the sea hare Dolabella auricularia [J]. Developmental & Comparative Immunology, 2003, 27(4): 305-311.
27 Mahoney MM. Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide [J]. FEBS Letters, 1995, 377(3): 519-522.
28 Buhimschi IA, Jabr M, Buhimschi C S, et al. The novel antimicrobial peptide [beta]3-defensin is produced by the amnion: A possible role of the fetal membranes in innate immunity of the amniotic cavity [J]. American Journal of Obstetrics and Gynecology, 2004, 191(5): 1678-1687.
29 Cole A M, Kim Y H, Tahk S, Hong T, et al. Calcitermin, a novel antimicrobial peptide isolated from human airway secretions [J]. FEBS Letters, 2001, 504(1-2): 5-10.
30 Wehkamp J, Harder J, Meissner B, et al. Human [beta]-defessin 3: A novel antimicrobial peptide preferentially expressed in ulcerative celitis [J]. Gastroenterology, 2001, 120(5, Supplement 1): A182-A182.
31 Masschalck B, van Houdt R, Michiels C W. High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin [J]. Int J Food Microbiolm, 2001, 64(3):325
32 Zhengrong Hao, Irene Kasumba, Serap Aksoy. Proventriculus(cardia) plays a crucial role in immunity in tsetse[J]. Insect Biochemistry and Molecular Biology, 2003, 33:11455-1164
33 Zasloff, M. Antimicrobial peptides of multicellular organisms[J]. Nature, 2002, 415:389-395
34 Jacopo Vizioli, Adam M, Richman, et al. The defensin peptide of the malaria vector mosquito Anopheles gambiae :antimicrobial activities and expression in adult mosquitoes [J]. Insect Biochemistry and Molecular Biology, 2001, 31:241-248
35 Tanaka K. P13-kinase p85 is a target molecular of praline-rich antimicrobial peptide to suppress proliferation of ras-transformed cells[J]. Jpn,J, Cancer Res. 2001, 92:959-967
36 Mare Cudie, Barry A, Condie et al. Development of novel antibacterial peptides that kill resistant isolates[J]. Pepides, 2002, 23:2071-2083
37 Lockey T D. Formation of pores in Escherichia coli cell membranes by a cecropinisolated from hemolymph of Heliothis virescens larvae[J]. Journal of biochemistry, 236(1): 263-271.
38 Juvvadi P, Antibacterial peptides cecropin A-melittin hybrid analogues, C-terminal amides [J]. Journal of Peptide Science, 1996, 2(4): 223-232.
39 Shai Y, Rapaport D, Gazit E, et al. Molecular mechanism of membrane permeation by cytolytic toxins [J]. Toxicon, 1995, 33(3): p. 272-272
40程家安,唐振华.昆虫分子科学[M].科学出版社, 2001. 77-87
41董占鹏.蚕类抗菌肽及其研究进展[J].蚕学通讯. 2003, 23(3): 14-21
42 Christensen B, Fink J, Merrifield R.B. Channel-forming properties of cecropins and related compounds incorporated into compounds into planar lipid membranes [J]. Proc Natl Acad Sci, USA, 1988, 85: 5072-5076.
43 Clague M J, Cherry R J. A comparative study of band 3 aggregation in erythrocyte membranes by melittin and other cationic agents [J]. Biochimica Biophysica Acta, 1989, 980: 93-99.
44周义文,尹一兵,涂植光等.家蝇抗菌肽抗菌活性及抗菌机制的初步研究[J].中国抗生素杂志, 2004, 29(5): 272-274
45 Hou L X, Shi Y H, Zhai P, et al. Inhibition of foodborne pathogens by Hf-1, a novel antibacterial peptide from the larvae of the housefly (Musca domestica) in medium and orange juice[J]. Food control, 2007, 18(11):1350-1357
46盛长忠,安春菊,耿华等.一种家蝇幼虫热稳定抗菌肽的分离纯化[J].南开大学学报(自然科学版), 2002, 35(4): 6-10
47邓小娟,黄自然.柞蚕抗菌肽的分离纯化及杀菌效应[J].华南农业大学学报(自然科学版), 2002, 23(3):13-16
48宫霞,施用晖,乐国伟.家蝇幼虫抗菌肽MDL-1的分离纯化及其对大肠杆菌超微结构的影响[J].昆虫学报, 2004, 47(1): 8-13
49陆婕,汪俊汉,钟雅等.弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究[J].微生物学报, 2006, 46(3): 406-411
50张虹,柳正良,王洪泉.大孔吸附树脂在药学领域的应用[J].中国医药工业杂志, 2001, 32(1):4l-44.
51李伯庭,王湘,李小进.大孔吸附树脂在天然产物分离中的应用[J].中草药, 1990, 21(8):42-44.
52刘琦英.大孔吸附树脂提取酸性蛋白酶抑制剂研究[J].离子交换与吸附, 1989, 5(4): 251-255
53何炳林,英文强.离子交换与吸附树脂[M].上海科技教育出版杜,1995
54 Silva E M, Pompeub D R, Larondelle Y, et al. Optimisation of the adsorption of polyphenols from Inga edulis leaves on macroporous resins using an experimental design methodology [J]. Separation purification technology, 2007, 43: 274-280
55 Wan J B, Zhang Q W, Ye W C, et al. Quantification and separation of protopanaxatriol and protopanaxadiol type saponins from Panax notoginseng with macroporous resins[J]. Separation purification technology, 2007, in press
56 Gao M, Huang W, Liu C Z. Separation of scutellarin from crude extracts of Erigeronbreviscapus (vant.) Hand. Mazz. by macroporous resins[J]. Journal of Chromatography B, 2007, 858: 22-26
57 Fu B Q, Liu J, Li H, et al. The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid[J]. Journal of Chromatography A, 2005, 1089: 18-24
58钱宝庭.离子交换树脂应用技术[M].天津科学技术出版杜, 1984. 302-354
59解芳,周任重,袁勤生.大孔吸附树脂纯化SOD [J].中国医药工业杂志, 1995, 26(I): 1-2
60刘琦英.大孔吸附树脂提取酸性蛋白酶抑制剂研究[J].离子交换与吸附, 1989, 5(4): 251-255.
61李从军,鲁明波,余龙江等.大孔吸附树脂纯化谷胱甘肽的初步研究[J].食品科技, 2006, 10:70-73
62赵利,王璋,许时婴.大孔吸附树脂对酪蛋白非磷肽吸附性的研究[J].食品工业科技, 2002, 12: 48-31
63 Andres E, Dimarcq J L. Letters to the Editor--Clinical development of antimicrobial peptides [J]. International Journal of Antimicrobial Agents, 2005, 25: 448–452
64 Hansen L T, Austin J W, Gill T A. Antibacterial effect of protamine in combination with EDTA and refrigeration [J]. International Journal of Food Microbiology, 2001, 66(3): 149-161.
65 Cintas L M, Casaus P, Fernandez M F, et al. Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria [J]. Food Microbiology, 1998, 15(3): 289-298.
66徐飞,施文,王启松等.柞蚕抗菌肽D基因的合成[J].科学通报, 1988, 21: 15-17.
67赵东红,戴祝英,周开亚等.中国家蚕抗菌肽人工合成类CMⅣ基因在甜菜夜蛾虫体中的表达[J].微生物学报, 2001, 41(06): 680-685
68 Prates M V, Sforca M L, Regis W C. The N MR-derived solution structure of an newcationic antimicrobial peptide from the skin secretion of the anuran Hyla Punctata[J]. J Biol Chem, 2004, 279(13):1308-1302
69 Sherman R A, Hall M J, T homas S. Medicinal maggots: an anciert remedy for some contemporary afflictions [J]. Annu Rev Entomol, \ 2000, 45: 55– 81
70 Sukontason K, Vogtsberger R C, Sukontason K L, et al. Surface ultrastructure of the third-instar larvae of Hydrotaea spinigera Stein (Diptera:Muscidae), a fly species of forensic importance [J]. J Vector Ecol, 2001, 26 (2):191-195.
71刘健敏,钟芳,麻建国.大豆生理活性肽的研究(Ⅰ)—酶法水解的工艺[J].无锡轻工大学学报, 2004, 23(3): 41-45.
72赵亚华,等.生物化学实验技术教程[M].广州:华南理工大学出版社, 2000.
73 Walker J M. Protein determination by UV absorption[J]. From: The protocols handbook. Humana Press Inc,Totowa, NJ: 3-5.
74牟德海, OPA柱前衍生反相高效液相色谱法测定氨基酸含量[J].色谱, 1997, 15(4): 319-321.
75 Jens A N. Enzymic Hydrolysis of food proteins [M]. Elsevier Applied Science Publishers, 1986, 6.
76 Ney K H. Voraussage der bitterkeit von peptiden aus deren Aminosaure zusammensetzung[J]. Z.Lebensm Untersuch Forsch, 1971, 147: 64-71.
77 Hoffmann D, Hultmark D, Boman H G. Insect immunity: Galleria mellonella and other lep idop tea have Cecrop in-P9-like factors active against gram-negative bacteria [J]. Insect Biochem, 1981, 11:5370-548
78 Owen R. Fennema,王璋等译.食品化学[M].北京:中国轻工业出版社, 2003
79曾宪明.无机盐对大孔吸附树脂吸附人参总皂苷的影响[J].中国医药工业杂志, 1992, 23(8): 339-342.
80郭立安.高效液相色谱法纯化蛋白质理论与技术[M].陕西科学技术出版社, 1993.
81 Boman H G, Hultmark D, Steriner H. Insect immunity. Purification and properties of three inducible bactericial proteins from hemolymph of immunized pupae of Hyalophora cecropia [J]. European Journal of Biochemistry, 1980, 106: 7-16.
82金冬雁,黎孟枫译, .J.萨姆布鲁克, E.F弗里寺, T.曼尼阿蒂斯著.分子克隆[M].第二版,北京:科学出版社, 1992, 9: 08-913
83张卓然.主编临床微生物学和微生物学检验[M].第三版,北京:人民卫生出版社, 2003, 492-500