CN105198979B - A kind of sea snake variant antibacterial peptide QHA1 and its preparation method and application - Google Patents

Abstract

The invention belongs to field of biomedicine technology, specifically a kind of chittue (Hydrophis cyanocinctus) antibacterial peptide QHA variant antibacterial peptides QHA1 and its preparation method and application.Variant antibacterial peptide is chittue (Hydrophis cyanocinctus) antibacterial peptide QHA variant QHA1, is straight-chain polypeptide, contains 17 amino acid residues, C-terminal amidation, molecular weight 2150.7Da, isoelectric point 12.32.The present invention relates to the antibacterial activities that variant antibacterial peptide QHA1 has broad-spectrum high efficacy, have the beneficial features such as molecular weight is small, simple in structure, hemolytic activity is low, preparation method is simple in addition.

Description

A kind of sea snake variant antibacterial peptide QHA1 and its preparation method and application

Technical field

The invention belongs to field of biomedicine technology, specifically a kind of chittue (Hydrophis Cyanocinctus) antibacterial peptide QHA variant antibacterial peptide QHA1 and its preparation method and application.

Background technology

The extensive abuse of conventional antibiotic leads to increasingly severe pathogenic microorganism resistance problems in recent years, gives the mankind Health care belt carrys out huge threat.Clinically the measure of reply drug-resistant microorganism infection is that use not yet used drug-resistant microorganism New or alternative antibiotic, therefore this antimicrobial agents for just needing Persisting exploitation new.

Antibacterial peptide is a kind of natural small molecule polypeptide of organism gene code, is that one kind of organism immune system is important Molecule all has direct killing effect to bacterium, fungi, virus even protozoon.Antibacterial peptide is small with molecular weight, structure is simple Single, the advantages that antibacterial activity is strong, bactericidal mechanism is unique, toxicity is low and is not easy to cause drug resistance, thus from self-discovery just by It is considered the antibiotic of new generation with very big potentiality to be exploited.Up to the present, it is found more than 1500 from different organisms A variety of different antibacterial peptides, and its number is also increasing.

Invention content

The purpose of the present invention is to provide a kind of chittue (Hydrophis cyanocinctus) antibacterial peptide QHA transformations Body antibacterial peptide QHA1 and its preparation method and application.

To achieve the above object, the technical solution adopted by the present invention is：

A kind of variant antibacterial peptide, variant antibacterial peptide are chittue (Hydrophis cyanocinctus) antibacterial peptide QHA variant QHA1, are straight-chain polypeptides, contain 17 amino acid residues, C-terminal amidation, molecular weight 2150.7Da, etc. Electricity point 12.32.

The variant antibacterial peptide is：Lys¹Phe²Phe³Lys⁴Arg⁵Leu⁶Leu⁷Lys⁸Ser⁹Val¹⁰Arg¹¹Arg¹² Ala¹³Val¹⁴Lys¹⁵Lys¹⁶Phe¹⁷-NH₂。

The application of variant antibacterial peptide, the variant antibacterial peptide be used to prepare antibacterials, inhibit bacterial growth drug, The purposes of preservative, animal feed or cosmetics precursor.

The beneficial effects of the present invention are：The present invention utilizes molecule according to the amino acid sequence of chittue antibacterial peptide QHA Remodeling method design improvement body QHA1, the variant have the antibacterial activity of broad-spectrum high efficacy, have that molecular weight is small, structure is simple in addition The beneficial features such as singly, hemolytic activity is low, preparation method is simple.

Specific implementation mode:

The essentiality content further illustrated the present invention below with embodiment, but present disclosure is not limited to This.

Embodiment 1

The chemical synthesis of sea snake variant antibacterial peptide QHA1

Chittue antibacterial peptide QHA is a kind of straight-chain polypeptide of gene code, contains 30 amino acid residues, molecular weight 3628.5Da, isoelectric point 12.61.Chittue antibacterial peptide QHA total orders are classified as： Lys¹Phe²Phe³Lys⁴Arg⁵Leu⁶Leu⁷Lys⁸Ser⁹Val¹⁰Arg¹¹Arg¹²Ala¹³Val¹⁴Lys¹⁵Lys¹⁶Phe¹⁷Arg¹⁸Lys¹⁹L ys²⁰Pro²¹Arg²²Leu²³Ile²⁴Gly²⁵Leu²⁶Ser²⁷Thr²⁸Leu²⁹Leu³⁰.According to the amino acid of chittue antibacterial peptide QHA Sequence is obtained variant QHA1 using the design of molecular modification method, and is carried out to it using the method for Solid-phase synthesis peptides Chemical synthesis, specific preparation method are as follows：

I, the preparation method of QHA1：According to the amino acid sequence of above-mentioned QHA1, with automatic Peptide synthesizer (433A, Applied Biosystems) its complete sequence is synthesized, utilize HPLC reversed phase column chromatography desalinations.

II, molecular weight determination uses Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF).

III, the QHA1 purified identifies that its purity, molecular weight determination use Matrix-assisted with high-efficient liquid phase chromatogram HPLC method Laser desorption ionization time of flight mass spectrometry (MALDI-TOF), isoelectric focusing electrophoresis measures isoelectric point, with automatic Protein Sequencer Measure amino acid sequence structure.

Measurement result is：

QHA1 is a kind of variant of chittue antibacterial peptide QHA.QHA1 is a kind of straight-chain polypeptide, contains 17 amino acid Residue, C-terminal amidation, molecular weight 2150.7Da, isoelectric point 12.32.QHA1 total orders are classified as： Lys¹Phe²Phe³Lys⁴Arg⁵Leu⁶Leu⁷Lys⁸Ser⁹Val¹⁰Arg¹¹Arg¹²Ala¹³Val¹⁴Lys¹⁵Lys¹⁶Phe¹⁷-NH₂。

Embodiment 2

QHA1 pharmacological evaluations：

1.QHA1 Determination of Antibacterial Activity：

(1) picking is stored in the test strain on inclined-plane and is spread evenly across MH solid mediums (purchased from Qingdao Hai Bo respectively Bioisystech Co., Ltd) on tablet, it will be placed in media surface by the filter paper of the 0.5cm diameters of sterilizing, dissolving is added dropwise In the 10 μ l of antibacterial peptide QHA1 sample solutions of the 2mg/ml of sterile deionized water, culture 18-20 hours, observation suppression are inverted in 37 DEG C Whether the formation of bacterium circle.If sample has antibacterial activity, Clear ＆ Transparent inhibition zone can be formed around filter paper, inhibition zone is got over Show that sample antibacterial activity is stronger greatly.

(2) (2 times dilute for antibacterial peptide QHA1 minimal inhibitory concentrations (Minimum Inhibitory Concentration) measurement Interpretation of the law)：

The bacterial strain with inhibition zone carries out MIC determination experiments in step experiment in selection.Test strain is inoculated into the training of MH liquid It supports in base (Qingdao Hai Bo Bioisystech Co., Ltd), 37 DEG C of shaken cultivations are then trained with fresh MH liquid to exponential phase Foster base will be cultivated to the culture solution of exponential phase and be diluted to 2 × 10⁵Cfu/ml is for use.

It is previously added 100 μ lMH fluid nutrient mediums in sterile each hole of 96 orifice plate, 100 μ l are then added in the first hole and use MH fluid nutrient mediums are diluted to the certain density antibacterial peptide QHA1 sample solutions through 0.22 μm of hole membrane filtration, are taken after mixing The 2nd hole is added in 100 μ l, successively doubling dilution (referring to table 1), and 100 μ l, which are sucked out, from the 9th hole discards, the 10th hole system control tube.

Table .1 dilution process

37 DEG C of slowly vibrating cultures 18 hours will be placed after above-mentioned each pipe mixing, and light absorption is measured at 600nm wavelength.Most Small Mlc is the minimum sample concentration of invisible bacterial growth.The results are shown in Table 2.

As can be seen from Table 2, antibacterial peptide QHA1 shows very by force gram-positive bacterium, gramnegative bacterium and fungi Antibacterial activity, including largely pathogenic bacteria are clinically separated, MIC value is in the range of 2.34-150 μ g/ml.

2 antibacterial peptide QHA1 antibacterial activities of table

Test strain	MIC(μg/ml)
		Escherichia coli ATCC25922	37.5
Escherichia coli KM	9.38
		Escherichia coli GZ	18.75
Shigella dysenteriae	2.34
		Klebsiella Pneumoniae 1400	18.75
Klebsiella oxytoca	150
		Proteus	37.5
Proteus mirabilis	4.69
		Stenotrophomonas maltophilia	150
Pseudomonas aeruginosa ATCC27853	150
		Pseudomonas aeruginosa ZY	9.38
Pseudomonas aeruginosa 1014	75
		Salmonella paratyphi A	9.38
Staphylococcus aureus 2706	9.38

Staphylococcus aureus 2810	150
		Staphylococcus aureus KM	2.34
Bacillus cereus	9.38
		Bacillus subtilis	9.38
Enterococcus faecium	9.38
		Nocadia	18.75
Candida albicans 2821	75
		Candida albicans 2710	150
Candida albicans 0102	9.38
		Candida glabrata	37.5
Ash group net slime mould	4.69

MIC：Minimal inhibitory concentration, result above are independently to repeat laboratory mean values three times.

2.QHA1 hemolytic activities measure：

The rabbit blood of acquisition is mixed into anti-freezing with Alsever's Solution, brine 2 times is simultaneously resuspended at 10⁷-10⁸Cell/ml's is outstanding Supernatant liquid.The good red cell suspension of above-mentioned dilution is mixed with the QHA1 samples for being dissolved in physiological saline, 37 DEG C of heat preservation 30min, then at 1000rpm centrifuges 5min, and supernatant surveys absorption value in 540nm.Negative control uses physiological saline, positive control to use TritonX-100, percent hemolysis calculate as follows：Percent hemolysis H%=A_Sample-A_{Negative control}/A_{Positive control}× 100%.Knot Fruit shows that sample concentration be the percent hemolysis of 100 μ g/ml, QHA1 is 2.9%, and in a concentration of 200 μ g/ml, QHA1's is molten Blood percentage is 3.98%, illustrates that QHA1 has lower hemolytic activity, is not easy to cause mammalian erythropoietin rupture dissolving.

Claims (3)

1. a kind of variant antibacterial peptide, it is characterised in that：The variant antibacterial peptide amino acid sequence is： Lys¹Phe²Phe³Lys⁴Arg⁵Leu⁶Leu⁷Lys⁸Ser⁹Val¹⁰Arg¹¹Arg¹²Ala¹³Val¹⁴Lys¹⁵Lys¹⁶Phe¹⁷-NH₂。

2. a kind of application of variant antibacterial peptide described in claim 1, it is characterised in that：The variant antibacterial peptide is for making Standby antibacterials, the purposes of preservative, animal feed or cosmetics precursor.

3. the application of variant antibacterial peptide according to claim 2, it is characterised in that：The antibacterials are to inhibit bacterium The drug of growth.