CN114196631A - Antibacterial functional polypeptide and preparation method and application thereof - Google Patents
Antibacterial functional polypeptide and preparation method and application thereof Download PDFInfo
- Publication number
- CN114196631A CN114196631A CN202010909862.1A CN202010909862A CN114196631A CN 114196631 A CN114196631 A CN 114196631A CN 202010909862 A CN202010909862 A CN 202010909862A CN 114196631 A CN114196631 A CN 114196631A
- Authority
- CN
- China
- Prior art keywords
- antibacterial
- preparation
- functional polypeptide
- culture
- polypeptide
- 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.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 55
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 55
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 48
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 238000013492 plasmid preparation Methods 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 17
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- 239000013612 plasmid Substances 0.000 claims abstract description 11
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 49
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 241000283690 Bos taurus Species 0.000 claims description 15
- 239000012190 activator Substances 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 15
- 210000000440 neutrophil Anatomy 0.000 claims description 15
- 239000002244 precipitate Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 11
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims description 11
- 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 claims description 10
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 10
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- 210000004027 cell Anatomy 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000009833 condensation Methods 0.000 claims description 10
- 230000005494 condensation Effects 0.000 claims description 10
- 238000003786 synthesis reaction Methods 0.000 claims description 10
- 239000012137 tryptone Substances 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 238000002386 leaching Methods 0.000 claims description 9
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007821 HATU Substances 0.000 claims description 5
- 241001052560 Thallis Species 0.000 claims description 5
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 239000006143 cell culture medium Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000004132 cross linking Methods 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000000178 monomer Substances 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 235000001014 amino acid Nutrition 0.000 description 12
- 108050004290 Cecropin Proteins 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 2
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 1
- 108010002069 Defensins Proteins 0.000 description 1
- 102000000541 Defensins Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108060003100 Magainin Proteins 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 241000269368 Xenopus laevis Species 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 108060001132 cathelicidin Proteins 0.000 description 1
- 102000014509 cathelicidin Human genes 0.000 description 1
- POIUWJQBRNEFGX-XAMSXPGMSA-N cathelicidin Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C1=CC=CC=C1 POIUWJQBRNEFGX-XAMSXPGMSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- Animal Behavior & Ethology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antibacterial functional polypeptide and a preparation method and application thereof, relates to the technical field of preparation methods and application of antibacterial functional polypeptides, and particularly relates to an antibacterial functional polypeptide and a preparation method and application thereof, wherein the antibacterial functional polypeptide comprises 2 or more than 2 disulfide bonds and antibacterial peptides with beta-folding structures; the preparation method of the antibacterial functional polypeptide comprises the following steps: s1, culturing bacterial colonies; s2, plasmid preparation liquid; s3, carrying out double enzyme digestion detection on the extracted recombinant plasmid; the antibacterial functional polypeptide is applied to the preparation of antibacterial drugs. The preparation method of the antibacterial functional polypeptide is simple, the related comparison tests are multiple, and the comparison test comparison data under the same condition are possessed, so that the beneficial effects caused by different antibacterial functional polypeptide contents under the comparison condition are judged, and the antibacterial functional polypeptide with different contents can be prepared into medicines, so that different application ranges can be adjusted.
Description
Technical Field
The invention relates to the technical field of antibacterial functional polypeptide, in particular to antibacterial functional polypeptide and a preparation method and application thereof.
Background
The antibacterial peptide antigen refers to a basic polypeptide substance with antibacterial activity generated by induction in an insect body, has a molecular weight of about 2000-7000 and consists of 20-60 amino acid residues. Most of the active polypeptides have the characteristics of strong alkalinity, heat stability, broad-spectrum antibiosis and the like. The first discovered antibacterial peptide in the world is a polypeptide with antibacterial activity produced by Swedish scientist G.Boman et al by injecting Neisseria monocytogenes and Escherichia coli to induce Xigubia silkworm pupae in 1980, and is named as Cecropins.
The antibacterial peptide can be divided into various types according to the structure, wherein Cathelicidin and Defensin are main types; they can be classified into 5 types, (1) single-chain cysteine residue-free α -helices, or peptides consisting of two α -helices joined by random coil; (2) antimicrobial peptides that are rich in certain amino acid residues but do not contain cysteine residues; (3) an antimicrobial polypeptide comprising 1 disulfide bond; (4) an antimicrobial peptide having 2 or more than 2 disulfide bonds and having a beta-sheet structure; (5) peptides with antibacterial activity derived from other larger polypeptides of known function. Among them, Cecropins, which were first isolated, Magainins, which were isolated from xenopus laevis, and the like, belong to the first class of antibacterial peptides, which are also commonly referred to as Cecropins, and research on such antibacterial peptides is currently being conducted.
The existing medicine preparation is generally prepared by extracting immune genes generated by plants or animals or peptides generated in animal cells, and has the defects of complicated operation steps, low precision and inaccurate comparison data.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an antibacterial functional polypeptide and a preparation method and application thereof, and solves the problems that the prior medicine preparation is generally used for extracting immune genes generated by plants or animals or preparing peptides generated in animal cells, the operation steps are complicated, the precision is low, and the comparison data is inaccurate.
In order to achieve the purpose, the invention is realized by the following technical scheme: an antibacterial functional polypeptide, its preparing process and application are disclosed, which contains 2 or more than 2 disulfide bonds and the antibacterial peptide with beta-folding structure.
Optionally, the preparation method of the antibacterial functional polypeptide comprises the following steps:
s1, culture colony: placing bovine neutrophil in a culture dish, adding yeast extract, tryptone and D-glucose, culturing, taking bovine neutrophil culture solution, transferring the bovine neutrophil culture solution into a cell culture medium, continuously culturing until a proper period of time, then placing the culture solution in a cell centrifuge, centrifuging, and collecting thalli;
s2, plasmid preparation liquid: pouring the culture bacterial liquid in the cultured bacterial colony obtained in the step S1 into an experimental shaker, filtering out a precipitate carried by the shaking liquid, adding cold ethanol into the supernatant of the centrifuge tube, slowly leaching the inner wall of the centrifuge tube, centrifuging, placing at room temperature to naturally dry the nucleic acid precipitate, dissolving the precipitate in TE buffer solution, and uniformly mixing in a mixer to obtain a plasmid preparation solution, wherein the prepared plasmid is used for the next experiment;
s3, taking the extracted recombinant plasmid for double enzyme digestion detection: according to the characteristics of the antibacterial peptide with 2 or more than 2 disulfide bonds and a beta-folded structure, the carboxyl of the next amino acid is activated by an activator through artificial synthesis, the activator is HBTU/HCTU/HITU/HATU + NMM/DIPEA or HOBT + DIC which is commonly used as the activator, the activated monomer reacts with free amino to form peptide bonds for crosslinking, a large amount of super-concentration reagents are used for driving the reaction to be finished at the step, and the reaction is repeatedly cycled until the synthesis is finished; after the amino acid condensation is finished, the amino acid condensation can be eluted by using an appropriate amount of TFA, and according to the acidity and basicity, a 10% TFA/DCM solution can be selected to be 100% TFA.
Optionally, 2-5% tryptone, 5-10% D-glucose, and 5-10% yeast extract are added to the cultured colonies in step S1.
Optionally, in the step S1, in the step of culturing the colonies, the colonies are cultured at 25 to 35 ℃ for 12 to 24 hours.
Optionally, in the step S1, culturing the colonies, 300-.
Optionally, in the step S1, in culturing the bacterial colony, the model of the cell centrifuge is AXTD4, the maximum rotation speed is 4000r/min, and the maximum relative centrifugal force is 2200 xg.
Optionally, in the step S2, in the plasmid preparation solution, the model of the experimental oscillator is LYZ-2102, the rotation frequency is 40-300 rpm, and the shimmy amplitude is phi 26 mm.
Optionally, in the step S2, 5-10ml of 70% cold ethanol is added to the plasmid preparation solution, and the inner wall of the centrifuge tube is slowly rinsed.
Optionally, in the step S2, in the plasmid preparation solution, the culture solution is centrifuged for 5-10 min.
Optionally, the antibacterial functional polypeptide is applied to preparation of antibacterial drugs.
The invention provides an antibacterial functional polypeptide and a preparation method and application thereof, and the antibacterial functional polypeptide has the following beneficial effects:
the preparation method of the antibacterial functional polypeptide is simple, related comparison tests are multiple, and comparison data of the comparison tests under the same condition are possessed, so that beneficial effects caused by different antibacterial functional polypeptide contents under the comparison condition are judged, and the antibacterial functional polypeptide with different contents can be prepared into medicines, so that different application ranges can be adjusted.
Detailed Description
In the following, technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Embodiment 1
The invention provides a technical scheme that: an antibacterial polypeptide with 2 or more than 2 disulfide bonds and beta-folded structure, its preparing process and its application are disclosed.
The preparation method of the antibacterial functional polypeptide comprises the following steps:
s1, culture colony: placing bovine neutrophil in a culture dish, adding yeast extract, tryptone and D-glucose, culturing, taking bovine neutrophil culture solution, transferring the bovine neutrophil culture solution into a cell culture medium, continuously culturing until a proper period of time, then placing the culture solution in a cell centrifuge, centrifuging, and collecting thalli;
s2, plasmid preparation liquid: pouring the culture bacterial liquid in the cultured bacterial colony obtained in the step S1 into an experimental shaker, filtering out a precipitate carried by the shaking liquid, adding cold ethanol into the supernatant of the centrifuge tube, slowly leaching the inner wall of the centrifuge tube, centrifuging, placing at room temperature to naturally dry the nucleic acid precipitate, dissolving the precipitate in TE buffer solution, and uniformly mixing in a mixer to obtain a plasmid preparation solution, wherein the prepared plasmid is used for the next experiment;
s3, taking the extracted recombinant plasmid for double enzyme digestion detection: according to the characteristics of the antibacterial peptide with 2 or more than 2 disulfide bonds and a beta-folded structure, the carboxyl of the next amino acid is activated by an activator through artificial synthesis, the activator is HBTU/HCTU/HITU/HATU + NMM/DIPEA or HOBT + DIC which is commonly used as the activator, the activated monomer reacts with free amino to form peptide bonds for crosslinking, a large amount of super-concentration reagents are used for driving the reaction to be finished at the step, and the reaction is repeatedly cycled until the synthesis is finished; after the amino acid condensation is finished, the amino acid condensation can be eluted by using an appropriate amount of TFA, and according to the acidity and basicity, a 10% TFA/DCM solution can be selected to be 100% TFA.
And step S1, adding 2% of tryptone, 5% of D-glucose and 5% of yeast extract into the cultured colonies.
In step S1, the colonies were cultured at 25 ℃ for 12 hours.
In step S1, among the cultured colonies, 300. mu.l of the culture medium was transferred to 500ml of the culture medium to continue the culture.
And step S1, culturing bacterial colony, wherein the model of the cell centrifuge is AXTD4, the maximum rotating speed is 4000r/min, and the maximum relative centrifugal force is 2200 Xg.
Step S2, in the plasmid preparation liquid, the model of the experimental oscillator is LYZ-2102, the rotation frequency is 40-300 rpm, and the shimmy amplitude is phi 26 mm.
Step S2, adding 570% cold ethanol into the plasmid preparation solution, and slowly leaching the inner wall of the centrifuge tube.
And step S2, in the plasmid preparation solution, centrifuging the culture solution for 5 min.
The application of the antibacterial functional polypeptide in preparing antibacterial drugs.
Example II
The invention provides a technical scheme that: an antibacterial polypeptide with 2 or more than 2 disulfide bonds and beta-folded structure, its preparing process and its application are disclosed.
The preparation method of the antibacterial functional polypeptide comprises the following steps:
s1, culture colony: placing bovine neutrophil in a culture dish, adding yeast extract, tryptone and D-glucose, culturing, taking bovine neutrophil culture solution, transferring the bovine neutrophil culture solution into a cell culture medium, continuously culturing until a proper period of time, then placing the culture solution in a cell centrifuge, centrifuging, and collecting thalli;
s2, plasmid preparation liquid: pouring the culture bacterial liquid in the cultured bacterial colony obtained in the step S1 into an experimental shaker, filtering out a precipitate carried by the shaking liquid, adding cold ethanol into the supernatant of the centrifuge tube, slowly leaching the inner wall of the centrifuge tube, centrifuging, placing at room temperature to naturally dry the nucleic acid precipitate, dissolving the precipitate in TE buffer solution, and uniformly mixing in a mixer to obtain a plasmid preparation solution, wherein the prepared plasmid is used for the next experiment;
s3, taking the extracted recombinant plasmid for double enzyme digestion detection: according to the characteristics of the antibacterial peptide with 2 or more than 2 disulfide bonds and a beta-folded structure, the carboxyl of the next amino acid is activated by an activator through artificial synthesis, the activator is HBTU/HCTU/HITU/HATU + NMM/DIPEA or HOBT + DIC which is commonly used as the activator, the activated monomer reacts with free amino to form peptide bonds for crosslinking, a large amount of super-concentration reagents are used for driving the reaction to be finished at the step, and the reaction is repeatedly cycled until the synthesis is finished; after the amino acid condensation is finished, the amino acid condensation can be eluted by using an appropriate amount of TFA, and according to the acidity and basicity, a 10% TFA/DCM solution can be selected to be 100% TFA.
Step S1, adding 3.5% tryptone, 7.5% D-glucose and 7.5% yeast extract into the cultured colony.
In step S1, the colonies were cultured at 30 ℃ for 18 hours.
In step S1, 400. mu.l of the cultured colonies were transferred to 500ml of a medium and cultured.
And step S1, culturing bacterial colony, wherein the model of the cell centrifuge is AXTD4, the maximum rotating speed is 4000r/min, and the maximum relative centrifugal force is 2200 Xg.
Step S2, in the plasmid preparation liquid, the model of the experimental oscillator is LYZ-2102, the rotation frequency is 40-300 rpm, and the shimmy amplitude is phi 26 mm.
Step S2, adding 7.5ml 70% cold ethanol into the plasmid preparation solution, and slowly leaching the inner wall of the centrifuge tube.
And step S2, in the plasmid preparation solution, centrifuging the culture solution for 7.5 min.
The application of the antibacterial functional polypeptide in preparing antibacterial drugs.
Example three
The invention provides a technical scheme that: an antibacterial polypeptide with 2 or more than 2 disulfide bonds and beta-folded structure, its preparing process and its application are disclosed.
The preparation method of the antibacterial functional polypeptide comprises the following steps:
s1, culture colony: placing bovine neutrophil in a culture dish, adding yeast extract, tryptone and D-glucose, culturing, taking bovine neutrophil culture solution, transferring the bovine neutrophil culture solution into a cell culture medium, continuously culturing until a proper period of time, then placing the culture solution in a cell centrifuge, centrifuging, and collecting thalli;
s2, plasmid preparation liquid: pouring the culture bacterial liquid in the cultured bacterial colony obtained in the step S1 into an experimental shaker, filtering out a precipitate carried by the shaking liquid, adding cold ethanol into the supernatant of the centrifuge tube, slowly leaching the inner wall of the centrifuge tube, centrifuging, placing at room temperature to naturally dry the nucleic acid precipitate, dissolving the precipitate in TE buffer solution, and uniformly mixing in a mixer to obtain a plasmid preparation solution, wherein the prepared plasmid is used for the next experiment;
s3, taking the extracted recombinant plasmid for double enzyme digestion detection: according to the characteristics of the antibacterial peptide with 2 or more than 2 disulfide bonds and a beta-folded structure, the carboxyl of the next amino acid is activated by an activator through artificial synthesis, the activator is HBTU/HCTU/HITU/HATU + NMM/DIPEA or HOBT + DIC which is commonly used as the activator, the activated monomer reacts with free amino to form peptide bonds for crosslinking, a large amount of super-concentration reagents are used for driving the reaction to be finished at the step, and the reaction is repeatedly cycled until the synthesis is finished; after the amino acid condensation is finished, the amino acid condensation can be eluted by using an appropriate amount of TFA, and according to the acidity and basicity, a 10% TFA/DCM solution can be selected to be 100% TFA.
And step S1, adding 5% of tryptone, 10% of D-glucose and 10% of yeast extract into the cultured colonies.
In the step S1, the colonies were cultured at 35 ℃ for 24 hours.
In step S1, 500. mu.l of the cultured colonies were transferred to 500ml of a medium and cultured.
And step S1, culturing bacterial colony, wherein the model of the cell centrifuge is AXTD4, the maximum rotating speed is 4000r/min, and the maximum relative centrifugal force is 2200 Xg.
Step S2, in the plasmid preparation liquid, the model of the experimental oscillator is LYZ-2102, the rotation frequency is 40-300 rpm, and the shimmy amplitude is phi 26 mm.
Step S2, adding 10ml of 70% cold ethanol into the plasmid preparation solution, and slowly leaching the inner wall of the centrifuge tube.
And step S2, in the plasmid preparation solution, centrifuging the culture solution for 10 min.
The application of the antibacterial functional polypeptide in preparing antibacterial drugs.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. An antibacterial functional polypeptide, which comprises 2 or more than 2 disulfide bonds and antibacterial peptide with a beta-folding structure.
2. An antibacterially functional polypeptide as claimed in claim 1, wherein: the preparation method of the antibacterial functional polypeptide comprises the following steps:
s1, culture colony: placing bovine neutrophil in a culture dish, adding yeast extract, tryptone and D-glucose, culturing, taking bovine neutrophil culture solution, transferring the bovine neutrophil culture solution into a cell culture medium, continuously culturing until a proper period of time, then placing the culture solution in a cell centrifuge, centrifuging, and collecting thalli;
s2, plasmid preparation liquid: pouring the culture bacterial liquid in the cultured bacterial colony obtained in the step S1 into an experimental shaker, filtering out a precipitate carried by the shaking liquid, adding cold ethanol into the supernatant of the centrifuge tube, slowly leaching the inner wall of the centrifuge tube, centrifuging, placing at room temperature to naturally dry the nucleic acid precipitate, dissolving the precipitate in TE buffer solution, and uniformly mixing in a mixer to obtain a plasmid preparation solution, wherein the prepared plasmid is used for the next experiment;
s3, taking the extracted recombinant plasmid for double enzyme digestion detection: according to the characteristics of the antibacterial peptide with 2 or more than 2 disulfide bonds and a beta-folded structure, the carboxyl of the next amino acid is activated by an activator through artificial synthesis, the activator is HBTU/HCTU/HITU/HATU + NMM/DIPEA or HOBT + DIC which is commonly used as the activator, the activated monomer reacts with free amino to form peptide bonds for crosslinking, a large amount of super-concentration reagents are used for driving the reaction to be finished at the step, and the reaction is repeatedly cycled until the synthesis is finished; after the amino acid condensation is finished, the amino acid condensation can be eluted by using an appropriate amount of TFA, and according to the acidity and basicity, a 10% TFA/DCM solution can be selected to be 100% TFA.
3. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: and step S1, adding 2-5% of tryptone, 5-10% of D-glucose and 5-10% of yeast extract into the cultured colonies.
4. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: in the step S1, the colonies are cultured at 25-35 ℃ for 12-24 h.
5. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: in the step S1, 500. mu.l of the culture colonies are transferred to 500ml of the culture medium for further culture.
6. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: in the step S1, in the colony culture, the model of the cell centrifuge is AXTD4, the maximum rotating speed is 4000r/min, and the maximum relative centrifugal force is 2200 Xg.
7. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: in the step S2, in the plasmid preparation solution, the model of the experimental oscillator is LYZ-2102, the rotation frequency is 40-300 rpm, and the shimmy amplitude is phi 26 mm.
8. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: and S2, adding 5-10ml of 70% cold ethanol into the plasmid preparation solution, and slowly leaching the inner wall of the centrifuge tube.
9. The method for preparing a polypeptide having antibacterial function according to claim 2, wherein: and S2, in the plasmid preparation solution, centrifuging the culture solution for 5-10 min.
10. An antibacterially functional polypeptide as claimed in any one of claims 1 to 9, wherein: the antibacterial functional polypeptide is applied to preparation of antibacterial drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010909862.1A CN114196631A (en) | 2020-09-02 | 2020-09-02 | Antibacterial functional polypeptide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010909862.1A CN114196631A (en) | 2020-09-02 | 2020-09-02 | Antibacterial functional polypeptide and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114196631A true CN114196631A (en) | 2022-03-18 |
Family
ID=80644358
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010909862.1A Pending CN114196631A (en) | 2020-09-02 | 2020-09-02 | Antibacterial functional polypeptide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114196631A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040136926A1 (en) * | 2002-11-08 | 2004-07-15 | Periathamby Antony Raj | Intra-oral drug delivery system |
| CN101775068A (en) * | 2009-01-14 | 2010-07-14 | 上海南方模式生物科技发展有限公司 | Novel natural antibacterial peptides, and coding sequence and uses thereof |
| CN102718844A (en) * | 2012-06-29 | 2012-10-10 | 兰州大学 | Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments |
| CN104017086A (en) * | 2014-06-24 | 2014-09-03 | 任建廷 | Fusion antimicrobial peptide and preparation method thereof |
| CN104478996A (en) * | 2014-12-08 | 2015-04-01 | 辽宁师范大学 | New cationic antimicrobial peptide and application thereof |
| CN105861522A (en) * | 2016-06-28 | 2016-08-17 | 江苏盐城源耀生物科技有限公司 | Antibacterial peptide Enterocin P and preparation method and application thereof |
| CN107344958A (en) * | 2017-03-31 | 2017-11-14 | 重庆理工大学 | Antibacterial pentapeptide derivative and its application |
-
2020
- 2020-09-02 CN CN202010909862.1A patent/CN114196631A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040136926A1 (en) * | 2002-11-08 | 2004-07-15 | Periathamby Antony Raj | Intra-oral drug delivery system |
| CN101775068A (en) * | 2009-01-14 | 2010-07-14 | 上海南方模式生物科技发展有限公司 | Novel natural antibacterial peptides, and coding sequence and uses thereof |
| CN102718844A (en) * | 2012-06-29 | 2012-10-10 | 兰州大学 | Antimicrobial peptide AMitP with acid activation property and synthesis and application thereof in preparation of anti-tumor medicaments |
| CN104017086A (en) * | 2014-06-24 | 2014-09-03 | 任建廷 | Fusion antimicrobial peptide and preparation method thereof |
| CN104478996A (en) * | 2014-12-08 | 2015-04-01 | 辽宁师范大学 | New cationic antimicrobial peptide and application thereof |
| CN105861522A (en) * | 2016-06-28 | 2016-08-17 | 江苏盐城源耀生物科技有限公司 | Antibacterial peptide Enterocin P and preparation method and application thereof |
| CN107344958A (en) * | 2017-03-31 | 2017-11-14 | 重庆理工大学 | Antibacterial pentapeptide derivative and its application |
Non-Patent Citations (3)
| Title |
|---|
| SUBBALAKSHMI C等: "Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin", 《FEBS LETTERS》, vol. 395, no. 1, pages 48 - 52, XP002053994, DOI: 10.1016/0014-5793(96)00996-9 * |
| 康静静等: "牛中性粒细胞β防御素5成熟肽的毕赤酵母表达及纯化", 《郑州牧业工程高等专科学校学报》, vol. 35, no. 1, pages 15 - 19 * |
| 王青等: "抗菌肽的特点及其基因工程研究进展", 《黑龙江畜牧兽医》, no. 8, pages 6 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113563463B (en) | Neutralizing nano antibody for resisting novel coronavirus SARS-CoV-2 and application thereof | |
| CN113717290B (en) | Composite transdermal recombinant fibronectin and application thereof | |
| CN113186109A (en) | Saccharomyces cerevisiae expression long-acting recombinant fibronectin and application thereof in cosmetics | |
| CN111349177B (en) | Preparation method and application of fusion antibacterial peptide CAT | |
| JPH03500050A (en) | Methods of reducing immunoglobulin E responses | |
| CN113880954B (en) | Recombinant human growth hormone and construction method and application thereof | |
| CN101514229B (en) | Human interferon alpha derivative and polyethylene glycol modified substance thereof | |
| CN114196631A (en) | Antibacterial functional polypeptide and preparation method and application thereof | |
| CN101280001B (en) | Preparation of human SDF-1 alpha, human SDF-1 alpha obtained therefrom and use thereof | |
| CN102020712A (en) | Human-like collagen for vaccine stabilizing agent and production method thereof | |
| CN115109795B (en) | Recombinant human-derived type III collagen injection and application thereof in skin collagen regeneration | |
| CN102898512B (en) | Recombinant plectasin as well as preparation method and application of recombinant plectasin | |
| CN114249814B (en) | Albumin HSA-hydrohopbic-IIIB with self-assembly performance and application thereof | |
| CN114591425B (en) | Specific antibody of new coronavirus S protein RBD region and preparation method and application thereof | |
| CN107722111A (en) | A kind of macrophages phagocytic capacity accelerator | |
| CN114591423B (en) | Specific antibody of new coronavirus N protein and preparation method and application thereof | |
| CN108864269B (en) | House dust mite allergen Der p 26 and preparation method and application thereof | |
| CN108892716B (en) | House dust mite allergen Der p29 and preparation method and application thereof | |
| CN112516288A (en) | Application of N-glycosylation modified ganoderma lucidum immunomodulatory protein | |
| CN109777807A (en) | A method of the localization and expression foreign protein in mitochondria | |
| CN109777808A (en) | A kind of polypeptide and its in nucleus and chloroplaset localization and expression foreign protein application | |
| CN114763379B (en) | Specific antibody of new coronavirus S protein, preparation method and application thereof | |
| CN108840917B (en) | House dust mite allergen Der p30 and preparation method and application thereof | |
| CN102628070A (en) | Method for converting expression vector of insulin precursor | |
| CN114409800B (en) | Method for preparing recombinant cystatin C |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220318 |
|
| RJ01 | Rejection of invention patent application after publication |