CN103788184A - Antibacterial peptide containing two leucine repeating units as well as preparation method and application - Google Patents
Antibacterial peptide containing two leucine repeating units as well as preparation method and application Download PDFInfo
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- CN103788184A CN103788184A CN201310715120.5A CN201310715120A CN103788184A CN 103788184 A CN103788184 A CN 103788184A CN 201310715120 A CN201310715120 A CN 201310715120A CN 103788184 A CN103788184 A CN 103788184A
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Abstract
本发明提供一种含有两个亮氨酸重复单元的抗菌肽及制备方法和应用，含有2个亮氨酸重复单元抗菌肽其氨基酸序列如SEQ No.1所示。本发明首先根据亮氨酸重复单元的基序和抗菌肽的氨基酸组成特点，得到亮氨酸重复单元的氨基酸序列，并将重复单元串联，以提高其肽链长度和净电荷数目；然后使用多肽合成仪，进行固相合成法合成多肽粗品；最后将合成多肽使用高效液相色谱进行纯化，并利用电喷雾质谱法进行鉴定。本发明的有益效果：(1)该多肽是根据生物体广泛存在的亮氨酸重复单元为基础而设计得到，具有广谱抗菌活性；(2)该多肽由22个氨基酸组成，肽链较短，制备方法和技术成熟，合成成本低。
The present invention provides an antibacterial peptide containing two leucine repeating units, its preparation method and application. The amino acid sequence of the antibacterial peptide containing two leucine repeating units is shown in SEQ No.1. The present invention first obtains the amino acid sequence of the leucine repeating unit according to the motif of the leucine repeating unit and the amino acid composition characteristics of the antimicrobial peptide, and connects the repeating units in series to increase the length of the peptide chain and the number of net charges; then use the polypeptide The synthesizer is used to synthesize crude peptides by solid-phase synthesis; finally, the synthesized peptides are purified by high-performance liquid chromatography and identified by electrospray mass spectrometry. Beneficial effects of the present invention: (1) The polypeptide is designed based on the leucine repeating unit widely present in organisms, and has broad-spectrum antibacterial activity; (2) The polypeptide consists of 22 amino acids and has a short peptide chain , the preparation method and technology are mature, and the synthesis cost is low.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of antibacterial peptide that contains two leucine repeating units and preparation method and application.
Background technology
Antibiotic application has improved human health and animal production efficiency.But the problem such as endurance strain, drug residue also occurs thereupon, is perplexing the mankind always.Owing to producing drug residue and bacterial drug resistance problem, therefore need find novel antibacterial preparation.Antimicrobial polypeptide wants immune integral part to have great development potentiality as body weight for humans, is the focus of researcher research.It is generally acknowledged, antibacterial peptide is the cytolemma that is used for destroying bacterium by physical adsorption etc., thereby kills bacterium, and therefore the probability of bacterium generation resistance is less.Antibacterial peptide is generally made up of 10 to 50 amino acid, and net charge is generally in+2～+ 9 scopes, and the ratio of hydrophobic amino acid can reach 50% or more.
The said structure of antibacterial peptide is similar to numerous protein composition motif with composition characteristic.Leucine repeating unit is extensively known for everybody institute, gains the name because it is rich in hydrophobic residue leucine.Leucine repeating unit is extensively present in exceeding in 60000 kinds of organism protein including animal, plant and microorganism.Wherein the motif of important leucine repeating unit is: a LxxLxLxxNxL, the protein that comprises these motifs can form spiral, various structure formation such as folding.Therefore, the present invention, using this motif as fundamental unit, by single leucine repeating unit and by the antibacterial peptide of 2 leucine repeating unit series connection, obtains brand-new antibacterial peptide thereby design by synthetic.
Summary of the invention
The object of the present invention is to provide a kind of antibacterial peptide that contains two leucine repeating units and preparation method and application, the present invention connects the antibacterial peptide with leucine repeating unit, thereby obtains brand-new antibacterial peptide, to improve its antibacterial effect.
Object of the present invention is achieved through the following technical solutions:
1, an antibacterial peptide that contains two leucine repeating units, sequence is as shown in SEQ No.1.
2, a kind of antibacterial peptide that contains two leucine repeating units as above, preparation method is as follows:
(1), according to the motif LxxLxLxxNxL of leucine repeating unit, hydrophobic residue and positive charge residue are enriched in motif, thereby obtain the aminoacid sequence (Leu) of leucine repeating unit
1, sequence is as shown in SEQ No.2; By this repeating unit series connection, to improve its peptide chain length and net charge number, obtain the antibacterial peptide (Leu) that contains two leucine repeating units
2, sequence is as shown in SEQ No.1;
(2) adopt solid state chemistry synthesis method, synthesize this two polypeptide crude products by Peptide synthesizer;
(3) use RPLC to carry out purifying synthetic polypeptide, and utilize electrospray mass spectrometry to identify synthetic polypeptide, complete the preparation of polypeptide.
3, a kind of antibacterial peptide that contains two leucine repeating units as above, the method for the mensuration minimal inhibitory concentration (MIC) that Standardization Institute of clinical labororatory of the employing U.S. is recommended, (Leu)
2minimal inhibitory concentration be 2-4 μ M, demonstrate the activity of very strong inhibition Gram-negative and positive bacteria growth.Thereby further provide for realizing the object of the invention the antibacterial peptide that contains two leucine repeating units, the application in preparation treatment gram-positive microorganism or gram positive bacterial infection disease medicament.
Beneficial effect of the present invention: (1) this polypeptide is that the leucine repeating unit extensively existing according to organism designs and obtains for basis, has broad spectrum antibiotic activity; (2) this polypeptide is made up of 22 amino acid, and peptide chain is shorter, preparation method and technology maturation, and synthetic cost is low.
Accompanying drawing explanation
Fig. 1 is antibacterial peptide (Leu)
1mass spectrum.
Fig. 2 is antibacterial peptide (Leu)
2mass spectrum.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1: the design of leucine repeating unit antibacterial peptide sequence
The motif of known leucine repeating unit is LxxLxLxxNxL, and wherein L is leucine, and x is arbitrary amino acid.The amino acid compositing characteristic of antibacterial peptide is mainly hydrophobicity and clean positive charge.Therefore, we enrich hydrophobic residue and positive charge residue in motif, thereby obtain the aminoacid sequence LeuArgArgLeuTrpLeuArgAlaAsnArgLeu of leucine repeating unit.Also by this repeating unit series connection, to improve its peptide chain length and net charge number, obtain two antibacterial peptide sequences simultaneously.As shown in table 1.
The aminoacid sequence of table 1 antibacterial peptide and molecular weight
Embodiment 2: leucine repeating unit antibacterial peptide sequence synthetic
Use Peptide synthesizer synthetic two polypeptide shown in table 1, select solid phase organic synthesis, adopt Fmoc protection synthesis method, compound direction holds N end to carry out one by one from C, and concrete steps are as follows:
(1) choose the Wang resin of having held first amino acid to be connected with C, be Fmoc-A (trt)-Wang (9-fluorenes methoxy carboxyl-trimethylammonium-A, wherein A is that C holds first amino acid), use dimethyl formamide (DMF) to soak 15min left and right to remove impurity; With the Fmoc protection on the DMF deresinate that contains 20% piperidines, reaction 20min, washing resin is until completely.Wash piperidines with DMF, remaining suspended solid is the A-Wang of deprotection.Check the quality of A-Wang deprotection with triketohydrindene hydrate detection agent.
(2) Fmoc-B (trt)-OH (9-fluorenes methoxy carboxyl-trimethylammonium-B, B is second amino acid of each antibacterial peptide C end) is carried out to condensation reaction with the Wang resin of deprotection obtained above; And then slough Fmoc group.Hold N end to extend one by one from C successively according to this program, until whole peptide chain is synthetic complete, when after last amino acid deprotection, with DMF washing 8 times, then use ethanol and methylene dichloride (DCM) cross washing 8 times.By trifluoroacetic acid (TFA): tri isopropyl chlorosilane (TIS): water=95: (volume ratio) mixed in 2.5: 2.5, preparation becomes at 20 ℃ of cutting reagent and polypeptide obtained above reacts 2h, and polypeptide is cut down from resin.Rotary Evaporators evaporation TFA, then add the precooling anhydrous diethyl ether of 10 times of left and right volumes to precipitate polypeptide 3h, separate out white powder solid.Vacuum-drying, obtains crude product polypeptide.
(3) above-mentioned crude product polypeptide is used 90% acetonitrile solution dissolve, use preparative chromatography post to carry out purifying, analysis mode chromatographic column detects purity.Semipreparative chromatography instrument is Waters Delta Prep4000, and preparative chromatography post is Waters X-Bridge C
i8, 5 μ m reversed-phase columns.Elutriant A is the aqueous solution that contains 0.1%TFA, and B is the acetonitrile solution that contains 0.1%TFA; Detect wavelength 220nm, the linear concentration gradient wash-out that type of elution is 30%B～65%B, flow velocity is 30mL/min.Collect purity higher than 95% cut, and lyophilize.Analysis mode high performance liquid chromatograph is Agilent1100, and analysis mode chromatographic column is SepaxGP-C
18reversed-phase column (4.6mm × 150mm, 5 μ m), the aqueous solution that elutriant A liquid is 0.1%TFA, B liquid is the acetonitrile solution containing 0.1%TFA; Detect wavelength 220nm.Type of elution is the linear concentration gradient wash-out of 50%B～75%B, and flow velocity is 1.0mL/min.
(4) Mass Spectrometric Identification of polypeptide: through electron spray mass spectrometry analysis, the molecular weight showing in mass spectrum is consistent with theoretical molecular by polypeptide obtained above.As depicted in figs. 1 and 2.Use high performance liquid chromatography to carry out purifying, make the purity of antibacterial peptide be greater than 95%.
Embodiment 3: the mensuration of antibacterial peptide anti-microbial activity
The method that adopts the mensuration minimal inhibitory concentration (MIC) of Standardization Institute of clinical labororatory of the U.S. (CLSI) recommendation, the while, for the feature of the cationic of antibacterial peptide, has been carried out corresponding improvement.Using 0.01% acetic acid (containing 0.2%BSA) as diluent, use doubling dilution to configure successively the antibacterial peptide solution of serial gradient.Get above-mentioned solution 50 μ L and be placed in 96 porocyte culture plates, then add respectively isopyknic bacterium liquid (～10 to be measured
5individual/mL) in each hole.Positive control (contain bacterium liquid and do not contain antibacterial peptide) and negative control (neither containing bacterium liquid also containing antibacterial peptide peptide) are set respectively.37 ℃ of constant temperature culture 20h, having no bottom, hole with naked eyes has the minimal inhibitory concentration that is of research of chaotic phenomenon.Result is as shown in table 2.As can be seen from Table 2, antibacterial peptide (Leu)
1minimal inhibitory concentration be 128 μ M or be greater than 128 μ M do not have or show very weak bacteriostatic activity; And (Leu)
2minimal inhibitory concentration be 2-4 μ M, demonstrate the activity of very strong inhibition Gram-negative and positive bacteria growth.
The bacteriostatic activity of table 2 antibacterial peptide
Claims (3)
1. an antibacterial peptide that contains two leucine repeating units, is characterized in that: sequence is as shown in SEQ No.1.
2. a kind of antibacterial peptide that contains two leucine repeating units according to claim 1, is characterized in that, preparation method is as follows:
(1), according to the motif LxxLxLxxNxL of leucine repeating unit, hydrophobic residue and positive charge residue are enriched in motif, thereby obtain the aminoacid sequence (Leu) of leucine repeating unit
1, sequence is as shown in SEQ No.2; By this repeating unit series connection, to improve its peptide chain length and net charge number, obtain the antibacterial peptide (Leu) that contains two leucine repeating units
2, sequence is as shown in SEQ No.1;
(2) adopt solid state chemistry synthesis method, synthesize this two polypeptide crude products by Peptide synthesizer;
(3) use RPLC to carry out purifying synthetic polypeptide, and utilize electrospray mass spectrometry to identify synthetic polypeptide, complete the preparation of polypeptide.
3. an application for a kind of antibacterial peptide that contains two leucine repeating units as claimed in claim 1, is characterized in that: the application in preparation treatment gram-positive microorganism or gram positive bacterial infection disease medicament.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106366162A (en) * | 2016-11-28 | 2017-02-01 | 东北农业大学 | Efficient alpha-helix antibacterial peptide GV and preparation method and application thereof |
| CN113056328A (en) * | 2018-11-23 | 2021-06-29 | 刘伟 | Microwave flow solid phase polypeptide synthesis method and system thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219831A (en) * | 2011-04-18 | 2011-10-19 | 江阴普莱医药生物技术有限公司 | Antibiotic peptide as well as preparation method and application thereof |
| CN102276691A (en) * | 2011-08-04 | 2011-12-14 | 东北农业大学 | Antibacterial peptides and preparation method thereof |
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- 2013-12-13 CN CN201310715120.5A patent/CN103788184A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219831A (en) * | 2011-04-18 | 2011-10-19 | 江阴普莱医药生物技术有限公司 | Antibiotic peptide as well as preparation method and application thereof |
| CN102276691A (en) * | 2011-08-04 | 2011-12-14 | 东北农业大学 | Antibacterial peptides and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Q.Q.MA等: "Rational design of cationic antimicrobial peptides by the tandem", 《AMINO ACIDS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106366162A (en) * | 2016-11-28 | 2017-02-01 | 东北农业大学 | Efficient alpha-helix antibacterial peptide GV and preparation method and application thereof |
| CN113056328A (en) * | 2018-11-23 | 2021-06-29 | 刘伟 | Microwave flow solid phase polypeptide synthesis method and system thereof |
| CN113056328B (en) * | 2018-11-23 | 2022-10-11 | 刘伟 | Microwave flowing solid phase polypeptide synthesis method and system thereof |
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Application publication date: 20140514 |