CN105273073A - Sea snake reforming body antibacterial peptide QHA2, preparation method and applications thereof - Google Patents
Sea snake reforming body antibacterial peptide QHA2, preparation method and applications thereof Download PDFInfo
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- CN105273073A CN105273073A CN201410236284.4A CN201410236284A CN105273073A CN 105273073 A CN105273073 A CN 105273073A CN 201410236284 A CN201410236284 A CN 201410236284A CN 105273073 A CN105273073 A CN 105273073A
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- antibacterial peptide
- qha2
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Abstract
The invention belongs to the technical field of biomedicine, and specifically relates to a Hydrophiscyanocinctus antibacterial peptide QHA reforming body antibacterial peptide QHA2, a preparation method and applications thereof. According to the present invention, the reforming body antibacterial peptide is the Hydrophiscyanocinctus antibacterial peptide QHA reforming body antibacterial peptide QHA2, is a straight-chain polypeptide, contains 18 amino acid residues, has an amidated C-terminal, has the molecular weight of 2306.9 Da, and has the isoelectric point of 12.49; and the reforming antibacterial peptide QHA2 has broad spectrum and efficient antibacterial activity, and further has beneficial characteristics of small molecular weight, simple structure, low hemolytic activity, simple preparation method, and the like.
Description
Technical field
The invention belongs to field of biomedicine technology, specifically a kind of chittue (Hydrophiscyanocinctus) antibacterial peptide QHA variant antibacterial peptide QHA2 and its preparation method and application.
Background technology
The extensive abuse of conventional antibiotic causes more and more serious pathogenic micro-organism resistance problems in recent years, brings huge threat to human health.Tackling the measure that drug-resistant microorganism infects clinically is use the not yet used newly or substituting microbiotic of drug-resistant microorganism, and therefore this just needs the antimicrobial agents that Persisting exploitation is new.
Antibacterial peptide is a kind of natural small molecule polypeptide of organism genes encoding, is a kind of important molecule of organism immune system, to bacterium, fungi, virus even protozoon all there is direct killing action.Antibacterial peptide has that molecular weight is little, structure is simple, anti-microbial activity is strong, bactericidal mechanism is unique, toxicity is low and not easily cause the advantages such as resistance, is therefore just considered to the microbiotic of new generation with very big potentiality to be exploited from self-discovery.Up to the present, from different organism, find the antibacterial peptide different more than kind more than 1500, and its number is also in increase.
Summary of the invention
The object of the present invention is to provide a kind of chittue (Hydrophiscyanocinctus) antibacterial peptide QHA variant antibacterial peptide QHA2 and its preparation method and application.
For achieving the above object, the technical solution used in the present invention is:
A kind of variant antibacterial peptide, variant antibacterial peptide is chittue (Hydrophiscyanocinctus) antibacterial peptide QHA variant QHA2, and it is straight-chain polypeptide, containing 18 amino-acid residues, its C-terminal amidation, molecular weight 2306.9Da, iso-electric point 12.49.
Described variant antibacterial peptide is: Phe
1phe
2lys
3arg
4leu
5leu
6lys
7ser
8val
9arg
10arg
11ala
12val
13lys
14lys
15phe
16lys
17arg
18-NH
2.
The application of variant antibacterial peptide, described variant antibacterial peptide is for the preparation of the purposes of antibacterials, bacteria growing inhibiting medicine, sanitas, animal-feed or makeup precursor.
Beneficial effect of the present invention is: the present invention is according to the aminoacid sequence of chittue antibacterial peptide QHA, utilize the variant QHA2 of molecular modification method design QHA, this variant has the anti-microbial activity of broad-spectrum high efficacy, has the beneficial features such as molecular weight is little, structure is simple, hemolytic activity is low, preparation method is simple in addition.
Embodiment:
Further illustrate essentiality content of the present invention by embodiment below, but content of the present invention is not limited thereto.
Embodiment 1
The chemosynthesis of sea snake variant antibacterial peptide QHA2
Chittue antibacterial peptide QHA is a kind of straight-chain polypeptide of genes encoding, containing 30 amino-acid residues, and molecular weight 3628.5Da, iso-electric point 12.61.Chittue antibacterial peptide QHA total order is classified as: Lys
1phe
2phe
3lys
4arg
5leu
6leu
7lys
8ser
9val
10arg
11arg
12ala
13val
14lys
15lys
16phe
17arg
18lys
19lys
20pro
21arg
22leu
23ile
24gly
25leu
26ser
27thr
28leu
29leu
30.According to the aminoacid sequence of chittue antibacterial peptide QHA, utilize molecular modification method design to obtain variant QHA2, and utilize the method for Solid-phase synthesis peptides to carry out chemosynthesis to it, concrete preparation method is as follows:
I, the preparation method of QHA2: according to the aminoacid sequence of above-mentioned QHA2, synthesizes its complete sequence with automatic Peptide synthesizer (433A, AppliedBiosystems), utilizes the desalination of HPLC reversed phase column chromatography.
II, molecular weight determination adopts Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF).
III, the QHA2 high-efficient liquid phase chromatogram HPLC method of purifying identifies its purity, molecular weight determination adopts Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF), isoelectric focusing electrophoresis measures iso-electric point, measures amino acid sequence structure with automatic Protein Sequencer.
Measurement result is:
QHA2 is a kind of variant of chittue antibacterial peptide QHA.QHA2 is a kind of straight-chain polypeptide, containing 18 amino-acid residues, and its C-terminal amidation, molecular weight 2306.9Da, iso-electric point 12.49.QHA2 total order is classified as: Phe
1phe
2lys
3arg
4leu
5leu
6lys
7ser
8val
9arg
10arg
11ala
12val
13lys
14lys
15phe
16lys
17arg
18-NH
2.
Embodiment 2
QHA2 pharmacological evaluation:
1.QHA2 Determination of Antibacterial Activity:
(1) the picking test strain be stored on inclined-plane is spread evenly across on MH solid medium (purchased from Qingdao Hai Bo Bioisystech Co., Ltd) flat board respectively, the filter paper of the 0.5cm diameter through sterilizing is placed in media surface, drip the antibacterial peptide QHA2 sample solution 10 μ l being dissolved in the 2mg/ml of sterilizing deionized water, be inverted in 37 DEG C and cultivate 18-20 hour, observe inhibition zone and whether formed.If sample has anti-microbial activity, then can form Clear & Transparent inhibition zone around filter paper, inhibition zone shows that more greatly sample anti-microbial activity is stronger.
(2) antibacterial peptide QHA2 minimal inhibitory concentration (MinimumInhibitoryConcentration) measures (2 times of dilution methods):
Selection has inhibition zone bacterial strain in step experiment carries out MIC determination experiment.Test strain is inoculated in MH liquid nutrient medium (Qingdao Hai Bo Bioisystech Co., Ltd), and the nutrient solution being cultured to logarithmic phase, to logarithmic phase, is then diluted to 2 × 10 with fresh MH liquid nutrient medium by 37 DEG C of shaking culture
5cfu/ml is stand-by.
100 μ lMH liquid nutrient mediums are added in advance in the aseptic 96 each holes of orifice plate, then add in the first hole 100 μ l MH liquid nutrient mediums be diluted to certain density through 0.22 μm of hole membrane filtration antibacterial peptide QHA2 sample solution, get 100 μ l after mixing and add the 2nd hole, doubling dilution (see table 1) successively, discard from the 9th hole sucking-off 100 μ l, the 10th hole system control tube.
Table .1 dilution process
Place 37 DEG C of slow shaking culture 18 hours by after above-mentioned each pipe mixing, measure photoabsorption in 600nm wavelength place.Minimal inhibitory concentration is cannot see the minimum sample concentration of bacterial growth.Result is as shown in table 2.
From table 2, antibacterial peptide QHA2 all shows very strong anti-microbial activity to gram positive bacterium, gram negative bacterium and fungi, and comprising a large amount of Clinical isolate bacterial, MIC value is in the scope of 2.34-150 μ g/ml.
Table 2 antibacterial peptide QHA2 anti-microbial activity
| Test strain | MIC(μg/ml) |
| Intestinal bacteria ATCC25922 | 37.5 |
| Intestinal bacteria KM | 18.75 |
| Intestinal bacteria GZ | 37.5 |
| Dysentery bacterium | 4.68 |
| Bacillus proteus | 150 |
| Proteus mirabilis | 18.75 |
| Pseudomonas aeruginosa ZY | 18.75 |
| Pseudomonas aeruginosa 1014 | 150 |
| Streptococcus aureus KM | 2.34 |
| Subtilis | 37.5 |
| Faecium | 18.75 |
| Nocadia | 9.38 |
| Candida albicans 2821 | 75 |
| Candida albicans 0102 | 9.38 |
| Candida glabrata | 18.75 |
| Ash group net slime mould | 9.38 |
MIC: minimal inhibitory concentration, above result is independently repeat laboratory mean values three times.
2.QHA2 hemolytic activity measures:
By the rabbit blood that gathers and A Shi liquid mixing anti-freezing, brine 2 times resuspended one-tenth 10
7-10
8 cethe suspension of ll/ml.The red cell suspension that above-mentioned dilution is good and the QHA2 sample mix being dissolved in physiological saline, 37 DEG C of insulation 30min, then in the centrifugal 5min of 1000rpm, supernatant liquor surveys absorption value in 540nm.Negative control uses physiological saline, and positive control uses TritonX-100, and percent hemolysis calculates as follows: percent hemolysis H%=A
sample-A
negative control/ A
positive control× 100%.Result shows that sample concentration be the percent hemolysis of 100 μ g/ml, QHA2 is 2.17%, and when concentration is 200 μ g/ml, the percent hemolysis of QHA2 is 1.45%, illustrates that QHA2 has lower hemolytic activity, not easily causes mammalian erythropoietin to break dissolving.
Claims (3)
1. a variant antibacterial peptide, is characterized in that: variant antibacterial peptide is chittue (Hydrophiscyanocinctus) antibacterial peptide QHA variant QHA2, and it is straight-chain polypeptide, containing 18 amino-acid residues, its C-terminal amidation, molecular weight 2306.9Da, iso-electric point 12.49.
2., by variant antibacterial peptide according to claim 1, it is characterized in that: described variant antibacterial peptide aminoacid sequence is: Phe
1phe
2lys
3arg
4leu
5leu
6lys
7ser
8val
9arg
10arg
11ala
12val
13lys
14lys
15phe
16lys
17arg
18-NH
2.
3. an application for variant antibacterial peptide according to claim 1, is characterized in that: described variant antibacterial peptide is for the preparation of the purposes of antibacterials, bacteria growing inhibiting medicine, sanitas, animal-feed or makeup precursor.
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| CN105273073B CN105273073B (en) | 2018-12-18 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001047535A2 (en) * | 1999-12-28 | 2001-07-05 | Oertenheim Bjoern | Pharmaceutical composition comprising snake venom and method for its manufacture |
| CN101412753A (en) * | 2008-09-27 | 2009-04-22 | 中国科学院昆明动物研究所 | Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof |
| CN102702333A (en) * | 2012-05-29 | 2012-10-03 | 中国药科大学 | Drug-resistant pathogen infection resistant polypeptide and uses thereof |
| CN103665111A (en) * | 2012-09-07 | 2014-03-26 | 山东国际生物科技园发展有限公司 | Transformed body HC-15 of antimicrobial peptide Hc-CATH (cathelicidins) of hydrophis cyanocinctus and preparation method and application of transformed body |
-
2014
- 2014-05-30 CN CN201410236284.4A patent/CN105273073B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001047535A2 (en) * | 1999-12-28 | 2001-07-05 | Oertenheim Bjoern | Pharmaceutical composition comprising snake venom and method for its manufacture |
| CN101412753A (en) * | 2008-09-27 | 2009-04-22 | 中国科学院昆明动物研究所 | Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof |
| CN102702333A (en) * | 2012-05-29 | 2012-10-03 | 中国药科大学 | Drug-resistant pathogen infection resistant polypeptide and uses thereof |
| CN103665111A (en) * | 2012-09-07 | 2014-03-26 | 山东国际生物科技园发展有限公司 | Transformed body HC-15 of antimicrobial peptide Hc-CATH (cathelicidins) of hydrophis cyanocinctus and preparation method and application of transformed body |
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| CN105273073B (en) | 2018-12-18 |
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