CN103360467A - Preparation method of broad-spectrum antibacterial polypeptide - Google Patents
Preparation method of broad-spectrum antibacterial polypeptide Download PDFInfo
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- CN103360467A CN103360467A CN2013102967664A CN201310296766A CN103360467A CN 103360467 A CN103360467 A CN 103360467A CN 2013102967664 A CN2013102967664 A CN 2013102967664A CN 201310296766 A CN201310296766 A CN 201310296766A CN 103360467 A CN103360467 A CN 103360467A
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- Prior art keywords
- polypeptide
- broad
- preparation
- antibacterial peptide
- lys
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- 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
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method of broad-spectrum antibacterial polypeptide. The preparation method comprises the following steps of: I, pre-measuring and screening; II, preparing antibacterial polypeptide; III, carrying out antibacterial detection; IV, carrying out hemolytic detection; V, preparing broad-spectrum antibacterial polypeptide; and finally, screening to obtain the polypeptide with the broad-spectrum antibacterial function. The polypeptide prepared from the method in the invention has a broad-spectrum antibacterial effect, particularly has a killing effect for streptococcus mutans and helicobacter pylori, and does not have a side effect for normal cells; moreover, the polypeptide is small in molecular weight, simple in structure, convenient for artificial synthesis, lasting in effect, free of haemolytic effect and capable of showing an important application value in treating bacterial infection, particularly treating streptococcus mutans and helicobacter pylori infection.
Description
Technical field
The present invention relates to a kind of preparation method of broad-spectrum antimicrobial polypeptide, belong to biological chemistry and biological technical field.
Background technology
Antibiotic resistance has become the serious problems that affect anti-infective therapy and animal husbandry development, and the research and development of new antimicrobial substance is the key that addresses this problem.Antibacterial peptide becomes the focus of research in recent years as a kind of important antimicrobial substance.Antibacterial peptide is a kind of small peptide with anti-microbial activity, is encoded, is induced generation through ambient conditions by the cell specific gene, extensively is present in organic sphere, is the important component part of natural immune system.Many antibacterial peptides also show antiviral, antineoplastic activity except anti-microbial effect is arranged.Antibacterial peptide generally is the cationic amphiphilic structure, can be specifically and electronegative Cell binding, and form aperture at cytolemma, or destroy membrane structure, cause necrocytosis.Its mechanism of action is unique, is difficult for producing resistance; At anti-tumor aspect, many antibacterial peptides have the function of selective killing tumour cell, and are especially lower to Mammals normal cell toxicity, are expected to become the modern weapon of human beat cancer, become one of focus of antitumor drug research and development.Many antibacterial peptides with anti-tumor activity have carried out clinical experiment, so antibacterial peptide has value of exploiting and utilizing.
Since the first antibacterial peptide cecropin in 1980 separates, many active polypeptide with similarity from bacterium, fungi, insect, batrachians, higher plant and Mammals, have in succession been found and have obtained.Along with the application of new technology, novel method, newfound antibacterial peptide quantity is also in sustainable growth.Traditional antibacterial peptide triage techniques is many to be separated step by step according to the protein separation method, and detects the anti-microbial activity of each component in the process of separating.Another thinking is to start with from separating antibacterial peptide gene, obtains antibacterial peptide by chemosynthesis or engineered method again.The screening of carrying out antibacterial peptide from the angle of gene isolation with separate, be the information of having utilized the structure and function of known antibacterial peptide, higher on efficient.Above method respectively has relative merits.Although kind and the preparation method of antibacterial peptide are a lot, but still need research and development to upgrade, have more the broad-spectrum antimicrobial polypeptide of using value.
Summary of the invention
The object of the invention is to solve the deficiency in the above-mentioned technology, provide a kind of have anti-multiple gram-positive microorganism, negative bacterium long-acting, heat-resisting, without the preparation method of the new broad-spectrum antimicrobial polypeptide of hemolytic.
For achieving the above object, the technology used in the present invention means are: a kind of preparation method of broad-spectrum antimicrobial polypeptide, and its step comprises:
One, prediction screening: download the genome sequence of Ascidian, searching wherein has the little open reading frame of potential encoding function, and these little open reading frame are translated respectively corresponding aminoacid sequence, therefrom screens antibacterial peptide;
Two, preparation antibacterial peptide: according to the antibacterial peptide sequence of prediction screening in the step 1, utilize the synthetic part of polypeptide wherein of solid-phase synthesis, and make the amidation of C end, the N end does not contain methionine(Met), the preparation antibacterial peptide, and carry out Mass Spectrometric Identification;
Three, germ resistance detects: it is 1mg/ml that antibacterial peptide is become concentration with dissolved in distilled water, adopts agar diffusion method to carry out anti-microbial activity and detects;
Four, hemolytic detects: utilize sheeps blood erythrocyte to detect the hemolytic action of antibacterial peptide;
Five, preparation broad-spectrum antimicrobial polypeptide: the polypeptide with broad-spectrum antimicrobial function that finishing screen is chosen.
Further, the bacterial strain that uses in the agar diffusion method in the described step 3 is the one or more combination of intestinal bacteria, staphylococcus aureus, tetrads, Candida albicans, Bacillus proteus, Pseudomonas aeruginosa, Streptococcus mutans, helicobacter pylori.
Further, the polypeptide for preparing in the described step 5, its sequence is: Phe Ser Leu Phe Phe Pro Tyr Ala Ala Leu Lys Trp Leu Arg Lys Leu Leu Lys Lys.
Further, the polypeptide for preparing in the described step 5 especially has lethal effect to Streptococcus mutans, helicobacter pylori.
Beneficial effect of the present invention is: the polypeptide of present method preparation has the anti-microbial effect of wide spectrum, especially Streptococcus mutans, helicobacter pylori is had lethal effect, and normal cell is free from side effects; Molecular weight is little, and is simple in structure, and synthetic is convenient; Effect is lasting, without hemolytic action, shows that it at the treatment bacterial infection, particularly has significant application value to the treatment aspect of Streptococcus mutans, helicobacter pylori infection.
Embodiment
Embodiment 1
A kind of preparation method of broad-spectrum antimicrobial polypeptide, its step comprises:
One, the information biology of antibacterial peptide screening
Take No. 2 chromogene groups of Ascidian sequence as template, the characteristics little according to the antibacterial peptide molecular weight at first utilize little open reading frame analysis software sORFfinder to screen the little open reading frame (sORFs) with potential encoding function from genome sequence.These sORFs are translated respectively corresponding aminoacid sequence, utilize antibacterial peptide forecasting software CAMP from sORFs, to screen antibacterial peptide.Predict altogether possibility and surpass 70% sequence more than 100 bars.
Two, the anti-microbial activity of polypeptide is identified
Select 8 possibilities to surpass the sequence of 90%, 2 possibility between 60-70%, entrust Shanghai Chu peptide biotech company to utilize solid-phase synthesis synthetic, and with the C-terminal amidation, synthetic by the HPLC purifying, purity reaches 96.68%, and identifies (table 1) through mass spectroscopy.
Table 1 synthetic peptide sequence table
| The polypeptide numbering | Sequence |
| P-01 | Phe Gly Thr Gly Val Lys Arg Lys Ala Gln Cys Asp Leu Lys Ser Lys |
| P -02 | Gly Lys Gly Leu Glu Val Ile Lys Trp Lys Leu Lys His Val Ile Gln Leu |
| P -03 | Leu Phe Ala Lys Ile Asn Gly Leu Lys Val Gly Pro Leu Lys Ile Gln Ile Val |
| P -04 | Phe?Ser?Leu Phe?Phe?Pro?Tyr Ala Ala Leu?Lys Trp Leu?Arg?Lys?Leu?Leu?Lys Lys |
| P -05 | Val Lys Leu Glu Ile Leu Gly Ser Lys Gly Gly Ala Lys Ile Glu Lys Val Ser Leu Ile Cys Leu Gln Phe Thr Ala Gly Tyr Lys |
| P -06 | Gly Ile Gly Arg Ile Gly Lys Val His Ala Ala Asn Leu Ile Pro Lys Gly |
| P -07 | Lys Lys Val Ile Ile Glu Tyr Val Lys Ile Glu Ala |
| P -08 | Val Lys Glu Glu Val Lys Phe Ser Lys Lys Trp |
| P -09 | Lys Phe Lys Leu Lys Ser Leu Lys Ile Ile Glu Tyr Lys Gln |
| P -10 | Val Ser Lys Ile Lys Lys Tyr Leu Lys Tyr Lys Asp Arg Ile |
Three, germ resistance detects:
It is 1mg/ml that polypeptide is become concentration with dissolved in distilled water, adopts agar diffusion method to carry out anti-microbial activity and detects.The bacterial strain that uses in the agar diffusion method is the one or more combination of intestinal bacteria, staphylococcus aureus, tetrads, Candida albicans, Bacillus proteus, Pseudomonas aeruginosa, Streptococcus mutans, helicobacter pylori, and the bacterial strain of these bacterial classifications is all available from Guangdong Province Institute of Micro-biology.After the bacterial strain recovery, be inoculated in 37 ℃ of incubated overnight of common LB substratum after, get respectively 50 μ l bacterium liquid and coat the LB solid medium.After leaving standstill the liquid absorption of carrying disease germs, adding respectively concentration is the polypeptide solution 10 μ l of 1mg/ml.Cultivate after 10 hours for 37 ℃, observe having or not and size of inhibition zone.Observe simultaneously inhibition zone and whether change in one week, judge antibacterial time length.The results are shown in Table 2.Wherein P-04 has broad spectrum antibiotic activity.
Table 2 polypeptide Analysis of Antimicrobial Activity result
+: anti-microbial activity is arranged;-: without antibiotic activity
Four, the minimum inhibitory concentration analysis of P-04 Antivariant streptococcus
Adopt blood agar plate to detect P-04 to the minimum inhibitory concentration of Streptococcus mutans.Leaving standstill half an hour after the bacterium liquid of 50 μ l activation coated blood agar plate, is 10,20,30,50 with 10 μ l concentration respectively, and the P-04 solution of 100,200,500,1000 μ g/ml adds culture dish, carries out mark.Cultivate after 24-48 hour, observe having or not of inhibition zone for 37 ℃.The result shows that minimum inhibitory concentration is 30 μ g/ml.
Five, the hemolytic of antibacterial peptide detects
Utilize sheeps blood erythrocyte to detect the hemolytic action of antibacterial peptide; Be that the antibacterial peptide solution 10 μ l of 1mg/ml drip on blood plate surface with concentration, do contrast with distilled water.Place 37 ℃ more than 24 hours, then observe having or not of haemolysis circle.The haemolysis circle does not all appear in all tested antibacterial peptides as a result.PRELIMINARY RESULTS shows that there is not hemolytic toxicity in described antibacterial peptide.
Preparation broad-spectrum antimicrobial polypeptide:
The polypeptide P-04 with broad-spectrum antimicrobial function that finishing screen is chosen, its sequence is: Phe Ser Leu Phe Phe Pro Tyr Ala Ala Leu Lys Trp Leu Arg Lys Leu Leu Lys Lys.
The polypeptide of present method preparation has the anti-microbial effect of wide spectrum, especially Streptococcus mutans, helicobacter pylori is had lethal effect, and normal cell is free from side effects; Molecular weight is little, and is simple in structure, and synthetic is convenient; Effect is lasting, without hemolytic action, shows that it at the treatment bacterial infection, particularly has significant application value to the treatment aspect of Streptococcus mutans, helicobacter pylori infection.
Be specific embodiments of the invention more than, its role is to content of the present invention is further described in detail, the reader is more readily understood, but does not consist of protection of the presently claimed invention.
Claims (4)
1. the preparation method of a broad-spectrum antimicrobial polypeptide is characterized in that, step comprises:
One, prediction screening: download the genome sequence of Ascidian, searching wherein has the little open reading frame of potential encoding function, and these little open reading frame are translated respectively corresponding aminoacid sequence, therefrom screens antibacterial peptide;
Two, preparation antibacterial peptide: according to the antibacterial peptide sequence of prediction screening in the step 1, utilize the synthetic part of polypeptide wherein of solid-phase synthesis, and make the amidation of C end, the N end does not contain methionine(Met), the preparation antibacterial peptide, and carry out Mass Spectrometric Identification;
Three, germ resistance detects: it is 1mg/ml that antibacterial peptide is become concentration with dissolved in distilled water, adopts agar diffusion method to carry out anti-microbial activity and detects;
Four, hemolytic detects: utilize sheeps blood erythrocyte to detect the hemolytic action of antibacterial peptide;
Five, preparation broad-spectrum antimicrobial polypeptide: the polypeptide with broad-spectrum antimicrobial function that finishing screen is chosen.
2. the preparation method of broad-spectrum antimicrobial polypeptide according to claim 1, it is characterized in that: the polypeptide for preparing in the described step 5, its sequence is: Phe Ser Leu Phe Phe Pro Tyr Ala Ala Leu Lys Trp Leu Arg Lys Leu Leu Lys Lys.
3. the preparation method of broad-spectrum antimicrobial polypeptide according to claim 1, it is characterized in that: the bacterial strain that uses in the agar diffusion method in the described step 3 is the one or more combination of intestinal bacteria, staphylococcus aureus, tetrads, Candida albicans, Bacillus proteus, Pseudomonas aeruginosa, Streptococcus mutans, helicobacter pylori.
4. the preparation method of broad-spectrum antimicrobial polypeptide according to claim 1, it is characterized in that: the polypeptide for preparing in the described step 5 is to Streptococcus mutans, helicobacter pylori tool lethal effect.
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| CN108434438A (en) * | 2018-06-22 | 2018-08-24 | 安徽科技学院 | Purposes and pharmaceutical composition of the antibacterial peptide in the drug for preparing treatment helicobacter pylori disease |
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