CN116731153B - 一种猪β-干扰素抗菌肽IFN7及其应用 - Google Patents
一种猪β-干扰素抗菌肽IFN7及其应用 Download PDFInfo
- Publication number
- CN116731153B CN116731153B CN202310988273.0A CN202310988273A CN116731153B CN 116731153 B CN116731153 B CN 116731153B CN 202310988273 A CN202310988273 A CN 202310988273A CN 116731153 B CN116731153 B CN 116731153B
- Authority
- CN
- China
- Prior art keywords
- ifn7
- peptide
- antibacterial peptide
- interferon
- antibacterial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 94
- 102000003996 Interferon-beta Human genes 0.000 title claims abstract description 29
- 108090000467 Interferon-beta Proteins 0.000 title claims abstract description 29
- 241000588724 Escherichia coli Species 0.000 claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 32
- 241000194017 Streptococcus Species 0.000 claims abstract description 21
- 230000003115 biocidal effect Effects 0.000 claims abstract description 20
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 14
- 239000003674 animal food additive Substances 0.000 claims abstract description 11
- 239000005452 food preservative Substances 0.000 claims abstract description 10
- 235000019249 food preservative Nutrition 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 6
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 13
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 13
- 230000002401 inhibitory effect Effects 0.000 claims description 10
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 10
- 239000004480 active ingredient Substances 0.000 claims description 7
- 229920001184 polypeptide Polymers 0.000 claims description 7
- 230000002147 killing effect Effects 0.000 claims description 6
- 239000003242 anti bacterial agent Substances 0.000 claims description 4
- 229940124350 antibacterial drug Drugs 0.000 claims description 4
- 239000004599 antimicrobial Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000000845 anti-microbial effect Effects 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 22
- 108020004414 DNA Proteins 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 12
- 210000000170 cell membrane Anatomy 0.000 description 11
- 230000000844 anti-bacterial effect Effects 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 108091005461 Nucleic proteins Proteins 0.000 description 5
- 241000282887 Suidae Species 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 108020000946 Bacterial DNA Proteins 0.000 description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 4
- 229960005542 ethidium bromide Drugs 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 206010040047 Sepsis Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 201000009906 Meningitis Diseases 0.000 description 2
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 2
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 230000002687 intercalation Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 208000013223 septicemia Diseases 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 125000003345 AMP group Chemical group 0.000 description 1
- 101100379209 Arabidopsis thaliana APD3 gene Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241000194021 Streptococcus suis Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000035472 Zoonoses Diseases 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 201000009052 suppurative lymphadenitis Diseases 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/565—IFN-beta
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Veterinary Medicine (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Communicable Diseases (AREA)
- Toxicology (AREA)
- Animal Husbandry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Nutrition Science (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种猪β‑干扰素抗菌肽IFN7,其氨基酸序列为YYLSILQYLK,抗菌肽IFN7的分子量为1303.56Da。实验证明,本发明抗菌肽IFN7对大肠杆菌、乙型链球菌能够产生了较强的抑制作用,可以用于制备治疗或预防由大肠杆菌、乙型链球菌引起疾病的药物、饲料添加剂,也可用于制备食品防腐剂。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种猪β-干扰素抗菌肽IFN7及其应用。
背景技术
大肠杆菌是世界范围内哺乳猪和断奶猪死亡的重要原因之一,是多种猪疾病的病原菌,可引起仔猪黄痢、白痢、水肿病,并可引起败血症、神经症状等。一般而言,在流行期间,由于缺乏被动保护,0~4日龄仔猪常发生大肠杆菌引起的新生仔猪腹泻。猪链球菌病是由不同群的链球菌引起的一种急性高热性人畜共患传染病。人类感染该病后,主要表现为败血症、脑膜炎、关节炎及化脓性***炎。猪感染后流行特点是仔猪以败血型和脑膜炎型为主,中、大猪以关节炎型为主,一年四季均可发病,传播迅速,死亡率高。
对猪大肠杆菌和链球菌病的治疗以抗生素为主,但近年来对猪源大肠杆菌和链球菌的抗药性严重,临床分离菌株多抗性较强,原有的抗生素药物已经无法达到杀灭致病菌的效果。因此寻找化学药物替代品也成为目前研究的一大热点。抗菌肽(AMPs)是指动物体内经诱导而产生的一类具有抗菌活性的碱性多肽物质,这类活性多肽多数具有强碱性、热稳定性以及广谱抗菌等特点。因此,抗菌肽被认为是抗生素的潜在替代品,在生物医药、饲料添加剂、食品防腐剂等领域有良好的应用前景。
干扰素是一类糖蛋白,它具有高度的种属特异性,负责保护宿主细胞免受病毒和细菌感染。有研究发现干扰素在抑菌过程中容易受到其它因素影响,从而导致抑菌活性不高。
因此,提供一种能有效抑制大肠杆菌和链球菌感染,同时具有良好稳定性的干扰素抗菌肽,具有重要的研究意义。
发明内容
本发明的目的在于克服现有技术的不足之处,提供了提供了一种猪β-干扰素抗菌肽IFN7及其应用,解决了上述背景技术中的问题。
本发明解决其技术问题所采用的技术方案之一是:提供猪β-干扰素抗菌肽IFN7。其氨基酸序列为YYLSILQYLK,如SEQIDNO:1所示。
以猪β-干扰素为目标寻找其序列中的抗菌肽存在可行的理论依据。因此,我们以猪β-干扰素为蛋白模版,分别采用糜蛋白酶和胰蛋白酶进行模拟酶解,获得多肽片段序列。然后利用APD3在线服务器根据抗菌肽的电荷性和疏水性对猪β-干扰素蛋白序列筛选,然后通过Swiss-model服务器对猪β-干扰素抗菌肽结构进行预测,发现了对大肠杆菌和乙型链球菌具有很强抗菌作用的抗菌肽序列YYLSILQYLK,命名为IFN7。
抗菌肽IFN7的分子量为1303.56 Da,所带电荷为+1,总疏水性比为40%。
抗菌肽IFN7可以从以下作用对细菌造成破坏:一方面,抗菌肽IFN7破坏细菌细胞膜,改变细菌细胞膜的通透性,并抑制细胞膜生成,导致细菌死亡;另一方面,抗菌肽IFN7与细菌基因组DNA相结合,抑制细菌DNA的合成,从而导致细菌死亡。
本发明解决其技术问题所采用的技术方案之二是:提供一种猪β-干扰素抗菌肽IFN7在制备抗菌药物中的应用,该抗菌药物用于抑制和/或杀灭大肠杆菌和乙型链球菌中的一种或多种。
本发明解决其技术问题所采用的技术方案之三是:提供一种抗菌药物,其有效成分包括猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
在本发明一较佳实施例中,所述抗菌药物的有效成分为猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
在本发明一较佳实施例中,所述抗菌药物用于抑制和/或杀灭大肠杆菌和乙型链球菌中的一种或多种。
本发明解决其技术问题所采用的技术方案之四是:提供一种饲料添加剂,其有效成分包括猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
在本发明一较佳实施例中,所述饲料添加剂的有效成分为猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
在本发明一较佳实施例中,所述饲料添加剂用于抑制和/或杀灭大肠杆菌和乙型链球菌中的一种或多种。
本发明解决其技术问题所采用的技术方案之五是:提供一种食品防腐剂,其有效成分为猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
在本发明一较佳实施例中,所述食品防腐剂用于抑制和/或杀灭大肠杆菌和乙型链球菌中的一种或多种。
本发明的抗菌肽可以采用本领域技术人员已知的方法合成,例如固相合成,并采用本领域技术人员已知的方法进行纯化,例如高效液相色谱法。
实施本发明,具有如下有益效果:
本发明以猪β-干扰素为研究对象,并通过筛选,发现一个全新氨基酸序列的多肽IFN7。研究IFN7对大肠杆菌和乙型链球菌的抑菌活性。实验结果表明,该肽对大肠杆菌和乙型链球菌具有强烈的抑制作用,它的抑菌机理是首先破坏细菌细胞膜结构,改变细胞膜的通透性,导致胞内物质发生渗漏,然后与细菌基因组DNA相结合,抑制细菌DNA的合成,从而导致细菌死亡。本发明抗菌肽IFN7可用于制备抗菌药物、饲料添加剂及食品防腐剂。
附图说明
图1为本发明抗菌肽IFN7的预测模型结构示意图。
图2为本发明抗菌肽IFN7对大肠杆菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0 μg/mL;
B:抗菌肽浓度250 μg/mL;
C:抗菌肽浓度125 μg/mL;
D:抗菌肽浓度62.5 μg/mL;
E:抗菌肽浓度31.25 μg/mL;
F:抗菌肽浓度15.6 μg/mL。
图3为本发明抗菌肽IFN7对乙型链球菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0 μg/mL;
B:抗菌肽浓度250 μg/mL;
C:抗菌肽浓度125 μg/mL;
D:抗菌肽浓度62.5 μg/mL;
E:抗菌肽浓度31.25 μg/mL;
F:抗菌肽浓度15.6 μg/mL。
图4为本发明抗菌肽IFN7对大肠杆菌时间-杀伤曲线(Time kill)图。在0.01M pH7.2磷酸盐缓冲液中稀释至104-5CFU/mL。抗菌肽浓度为31.25 μg/mL。
图5为本发明抗菌肽IFN7对乙型链球菌时间-杀伤曲线(Time kill)图。在0.01MpH 7.2磷酸盐缓冲液中稀释至104-5CFU/mL。抗菌肽浓度为31.25 μg/mL。
图6为本发明抗菌肽IFN7与大肠杆菌透射电镜图,其中A为大肠杆菌空白对照;B为抗菌肽IFN7处理后的大肠杆菌组。
图7为本发明抗菌肽IFN7与大肠杆菌DNA结合电泳图,其中,条带5:空白对照;条带1-4:分别是IFN7与DNA质量比为25/4,25/2,25/1,50/1。
图8为本发明抗菌肽IFN7竞争性结合大肠杆菌DNA的荧光光谱图。抗菌肽浓度为15.6 μg/mL、31.3 μg/mL、62.5 μg/mL。
图9为本发明抗菌肽IFN7对大肠杆菌细胞膜内还原糖泄露的测定图。抗菌肽浓度为31.25 μg/mL、62.5 μg/mL。
图10为本发明抗菌肽IFN7对大肠杆菌细胞膜内核酸泄露的测定图。抗菌肽浓度为31.25 μg/mL、62.5 μg/mL、125 μg/mL。
图11为本发明抗菌肽IFN7对大肠杆菌细胞膜内蛋白质泄露的测定图。抗菌肽浓度为31.25 μg/mL、62.5 μg/mL、125 μg/mL。
具体实施方式
为了更好地理解本发明,下面结合实施例和附图对本发明做进一步的详细说明,但本领域技术人员了解,下述实施例不是对本发明保护范围的限制,任何在本发明基础上做出的改变和变化,都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:猪β-干扰素抗菌肽的筛选
在National Center for Biotechnology Information网站获取所需的猪β-干扰素的蛋白序列,利用在线软件Expasy对β-干扰素进行糜蛋白酶和胰蛋白酶模拟消化酶解,结合在线软件APD3进行疏水率及电荷数的计算,以此评估肽段的抗菌效力,筛选β-干扰素的肽段序列。然后通过Swiss-model服务器对猪β-干扰素抗菌肽IFN7的结构进行预测,并利用Pymol软件进行编辑和修改,得到抗菌肽IFN7的二级结构,如图1所示。
实施例2:抗菌肽IFN7的最低抑制浓度(MIC)测定
将大肠杆菌和乙型链球菌分别在37℃培养12 h至对数生长期,在0.01 M pH 7.2磷酸盐缓冲液中稀释至104-5CFU/mL。将抗菌肽IFN7溶于磷酸盐缓冲中,37℃等体积与菌混合后培养2 h。最低抑菌浓度(MIC)是指在37℃培养过夜后,能够抑制细菌生长的抗菌肽最低浓度。如图2和图3所示,IFN7对大肠杆菌和乙型链球菌的最低抑菌浓度(MIC)为31.25 μg/mL。
实施例3:抗菌肽IFN7的时间-杀伤曲线(Time-Kill曲线)测定
使用平板菌落计数法测定抗菌肽IFN7的时间-杀菌曲线。取-20℃保存的大肠杆菌和乙型链球菌在37℃培养12 h至对数生长期,在0.01 M pH 7.2磷酸盐缓冲液中稀释至104 -5CFU/mL。将抗菌肽IFN7溶于磷酸盐缓冲中,并稀释至62.5 μg/mL、125 μg/mL,分别与菌液等体积混合,于37℃恒温孵育。在不同的时间点(即0.5、1、1.5、2、2.5和3h)取20μL细菌悬液均匀涂在营养肉汤平板上,于37℃的培养24 h后计数菌落。如图4和图5所示,抗菌肽IFN7对大肠杆菌和乙型链球菌在4h左右具有明显杀灭效果,且随着作用时间增加细菌没有再进行生长繁殖。
实施例4:抗菌肽IFN7对大肠杆菌的透射电子显微镜观察
将500 µL对数生长期大肠杆菌菌液加入等体积抗菌肽IFN7(终浓度62.5 μg/mL),在37℃培养2 h。培养结束后于2700 g离心2 min,收集菌体沉淀用PBS洗涤3次。使用2.5%戊二醛固定12 h。使用乙醇(30%-100%)脱水,再用丙酮处理20 min后,将样品在70 ℃下24 h,制备70-90 nm的薄片,使用柠檬酸铅和乙酸铀酯染色后观察。如图6所示,空白组大肠杆菌细胞膜完整,界限清晰,而抗菌肽IFN7组的细菌细胞膜边界模糊,细胞质密度降低,出现膜壁分离的情况,证实抗菌肽IFN7对大肠杆菌的细胞膜具有一定的破坏作用,从而影响细菌的正常生长。
实施例5:抗菌肽IFN7与细菌DNA的相互作用
采用DNA凝胶阻滞法研究了抗菌肽IFN7与大肠杆菌基因组DNA的相互作用。将大肠杆菌分别在37℃的50mL营养肉汤培养基(NB)中培养12h,用细菌基因组DNA提取试剂盒提取细菌基因组DNA,使用超微量分光光度计测测定所提取DNA的浓度,并通过测定260 nm和280nm处的光密度比(OD260/OD280≥1.9)判断所提取DNA的纯度。接下来,将10μL DNA(198 ng/μL)与质量比为25/4,25/2,25/1,50/1抗菌肽IFN7在25℃下混合90 min,将混合物在0.8%琼脂糖凝胶上进行电泳。使用GelDocXR凝胶成像***(Bio-Rad,美国)在紫外线照射下观察DNA凝胶变化,如图7所示。随着抗菌肽IFN7的浓度增加,DNA条带亮度明显降低,表明抗菌肽IFN7参与了大肠杆菌DNA及其结合蛋白的降解作用。
实施例6:抗菌肽IFN7竞争性结合DNA的荧光光谱实验
通过测定DNA-EB复合物体系与竞争物(抗菌肽IFN7)作用的荧光光谱的变化,来判定抗菌肽IFN7是否同EB一样通过嵌插方式与DNA结合。首先,用1×TE缓冲液将制备的菌体DNA稀释为50 μg/mL。然后,在96孔板的每个孔中加入5 μL的DNA溶液和10 μL 100 μg/mL的溴化乙锭(EB)溶液,混匀后置于37℃避光孵育10 min。接着加入50 μL不同浓度的抗菌肽IFN7,使最终抗菌肽IFN7的浓度分别为15.6 μg/mL、31.25 μg/mL、62.5 μg/mL,对照组为50μL 0.01 M PBS(pH 7.2)。混匀后置于37℃生化培养箱中避光孵育30 min,孵育结束后,用多功能酶标仪测定样品在激发波长535 nm及发射波长570 nm-710 nm范围内的荧光光谱。由图8可知,随着抗菌肽IFN7浓度的增加,EB-DNA复合物的荧光强度降低,说明抗菌肽IFN7将之前结合在DNA碱基对上的EB以嵌插方式替代下来,使整个体系的荧光强度降低。
实施例7:抗菌肽IFN7对细菌胞内成分泄露的影响
将大肠杆菌接种于37℃的20mL营养肉汤培养基(NB)中培养12 h使其生长到对数期。用0.1 M PBS 将离心后的菌体清洗三次后调节其浓度为104-5CFU/mL。将抗菌肽IFN7溶于0.1 M PBS中,使其浓度为31.25 μg/mL、62.5 μg/mL,空白对照组用0.1 M PBS替换,37℃等体积与菌混合孵育培养,在培养0、30、60、90 min后分别取出100 μL菌液离心取上清液,采用DNS比色法利用多功能酶标仪测量在540 nm下的吸光度,并与葡萄糖标准曲线进行比较。由图9可知,空白组大肠杆菌菌液中还原糖含量较低,抗菌肽IFN7处理组的菌液中还原糖渗出量明显增加,且随着抗菌肽IFN7浓度增加,还原糖渗出量也随之增加。
将大肠杆菌接种于20 mL营养肉汤培养基(NB)中培养12 h使其生长到对数期。用0.1M PBS 将离心后的菌体清洗三次后调节其浓度为104-5CFU/mL。将抗菌肽IFN7溶于0.1 MPBS中,使其浓度为31.25 μg/mL、62.5 μg/mL、125 μg/mL,空白对照组用0.1 M PBS替换,等体积与菌混合,利用多功能酶标仪测定不同时间点在260 nm、280 nm下的吸光度,从而计算菌体核酸和蛋白质的渗出量。从图10和图11可知,空白组大肠杆菌菌液核酸和蛋白质的含量较低,但抗菌肽IFN7处理组菌液核酸和蛋白质渗出量明显增多,且随着抗菌肽IFN7浓度增加,菌液中核酸和蛋白质渗透量增加,60 min后,抗菌肽IFN7处理组菌液核酸和蛋白质渗透量基本保持不变。
综上,本发明提供了一种全新的抗菌肽IFN7对大肠杆菌和乙型链球菌的最低抑菌浓度MIC均为31.25 μg/mL,能够抑制大肠杆菌和乙型链球菌生长。本发明抗菌肽IFN7首先穿透细菌的细胞膜,使得细胞质密度降低、膜壁分离,然后与细菌基因组DNA相结合,抑制细菌DNA的合成,从而导致细菌死亡。因此,本发明抗菌肽IFN7可用于制备抗菌药物、饲料添加剂或食品防腐剂。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
Claims (9)
1. 一种猪β-干扰素抗菌肽IFN7,其氨基酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的猪β-干扰素抗菌肽IFN7在制备抗菌药物中的应用,其特征在于:所述抗菌药物用于抑制和/或杀灭大肠杆菌、乙型链球菌中的一种或多种。
3. 一种抗菌药物,其特征在于:其有效成分包括猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
4. 如权利要求3所述的抗菌药物,其特征在于:其有效成分为猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
5. 一种饲料添加剂,其特征在于:其有效成分包括猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
6. 如权利要求5所述的饲料添加剂,其特征在于:其有效成分为猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
7.如权利要求5或6任一项所述的饲料添加剂,其特征在于:所述饲料添加剂用于抑制和/或杀灭大肠杆菌、乙型链球菌中的一种或多种。
8. 一种食品防腐剂,其特征在于:其有效成分为猪β-干扰素抗菌肽IFN7,所述抗菌肽IFN7的氨基酸序列为SEQ ID NO:1。
9.如权利要求8所述的一种食品防腐剂,其特征在于:所述食品防腐剂用于抑制和/或杀灭大肠杆菌、乙型链球菌中的一种或多种。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310988273.0A CN116731153B (zh) | 2023-08-08 | 2023-08-08 | 一种猪β-干扰素抗菌肽IFN7及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310988273.0A CN116731153B (zh) | 2023-08-08 | 2023-08-08 | 一种猪β-干扰素抗菌肽IFN7及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116731153A CN116731153A (zh) | 2023-09-12 |
CN116731153B true CN116731153B (zh) | 2023-11-07 |
Family
ID=87915344
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310988273.0A Active CN116731153B (zh) | 2023-08-08 | 2023-08-08 | 一种猪β-干扰素抗菌肽IFN7及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116731153B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117700486B (zh) * | 2024-02-05 | 2024-04-19 | 广州爱保农生物科技有限公司 | 一种乙酰短杆菌发酵液抗菌肽yx-2及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103570836A (zh) * | 2013-10-25 | 2014-02-12 | 江苏众红生物工程创药研究院有限公司 | 一种重组猪干扰素β1-Fc融合蛋白及其编码基因和表达方法 |
CN110357971A (zh) * | 2019-08-21 | 2019-10-22 | 中国科学院微生物研究所 | 一种用于紧急预防非洲猪瘟的猪复合干扰素 |
CN115724943A (zh) * | 2022-08-05 | 2023-03-03 | 广州爱保农生物科技有限公司 | 一种大黄鱼胶原蛋白抗菌肽lp-p1及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8936782B2 (en) * | 2011-11-22 | 2015-01-20 | Cedars-Sinai Medical Center | Interferon beta as antibacterial agents |
CN108752457B (zh) * | 2018-05-23 | 2019-06-21 | 华中农业大学 | 一种来源于草鱼干扰素的衍生多肽及其应用 |
-
2023
- 2023-08-08 CN CN202310988273.0A patent/CN116731153B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103570836A (zh) * | 2013-10-25 | 2014-02-12 | 江苏众红生物工程创药研究院有限公司 | 一种重组猪干扰素β1-Fc融合蛋白及其编码基因和表达方法 |
CN110357971A (zh) * | 2019-08-21 | 2019-10-22 | 中国科学院微生物研究所 | 一种用于紧急预防非洲猪瘟的猪复合干扰素 |
CN115724943A (zh) * | 2022-08-05 | 2023-03-03 | 广州爱保农生物科技有限公司 | 一种大黄鱼胶原蛋白抗菌肽lp-p1及其应用 |
Non-Patent Citations (1)
Title |
---|
Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens;Jenni N. Weeks-Gorospe等;Journal of Virology;第86卷(第17期);全文 * |
Also Published As
Publication number | Publication date |
---|---|
CN116731153A (zh) | 2023-09-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN116731153B (zh) | 一种猪β-干扰素抗菌肽IFN7及其应用 | |
Quesada-Moraga et al. | Intra-specific variation in virulence and in vitro production of macromolecular toxins active against locust among Beauveria bassiana strains and effects of in vivo and in vitro passage on these factors | |
Guo-Ping et al. | Overview of silkworm pathology in China | |
Nowruzi et al. | Production of the neurotoxin homoanatoxin-a and detection of a biosynthetic gene cluster sequence (anaC) from an Iranian isolate of Anabaena | |
CN113461777B (zh) | 一种具有抗真菌作用的抗菌肽及其制备方法和应用 | |
CN113321708B (zh) | 一种人工设计抗菌肽的制备及其在水产上的应用 | |
CN113151069B (zh) | 一种枯草芽孢杆菌及其在制备抗菌肽、饲料中的应用 | |
CN112746044A (zh) | 一株植物乳杆菌突变菌株及其在苜蓿青贮中的应用 | |
CN117164665A (zh) | 一种人鼻液α-2-巨球蛋白抗菌肽A2M3及其应用 | |
Lodeiro et al. | Early interactions of Bradyrhizobium japonicum and soybean roots: specificity in the process of adsorption | |
CN115724943A (zh) | 一种大黄鱼胶原蛋白抗菌肽lp-p1及其应用 | |
CN113087771B (zh) | 一种南美白对虾dna结合抗菌肽vpdb40及其应用 | |
Bajpai et al. | Physiological evidence indicates microcystin-LR to be a part of quantitative chemical defense system | |
CN113429469A (zh) | 一种家蚕抗菌肽BMGlvA2重组蛋白制备方法及其应用 | |
CN109627285B (zh) | 迦得拟微绿球藻抗菌肽及其应用 | |
CN117700486B (zh) | 一种乙酰短杆菌发酵液抗菌肽yx-2及其应用 | |
Puar et al. | The biosynthesis of hazimicins: possible origin of isonitrile carbon | |
CN117164669A (zh) | 一种凡纳滨对虾延伸因子抗菌肽bce3及其应用 | |
CN117986327B (zh) | 一种抗菌肽rs12及其应用 | |
CN118047841B (zh) | 一种发酵桑叶抗菌肽Squ8及其应用 | |
Shanmugaraju et al. | Partial purification and characterization of Anti-MRSA peptide from marine Pseudomonas aeruginosa | |
CN115710305A (zh) | 一种发酵大黄鱼琥珀酸脱氢蛋白抗菌肽sdh73及其应用 | |
CN116622670A (zh) | 一种蜡样芽孢杆菌ATP合酶β亚基抗菌肽BaD9及其应用 | |
Thankaraj et al. | Proteomic approaches to identify resistance proteins from Rhizoctonia solani infected rice, treated with seaweed and bioinoculants | |
CN115894661B (zh) | 一种抗菌肽及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |