阅读说明：本技术 一种人肠道防御素5衍生线性多肽及其制备方法和应用 (Human intestinal defensin 5 derived linear polypeptide and preparation method and application thereof ) 是由 王成 王军平 赵高梅 陈银 韩松伶 于 2019-10-18 设计创作，主要内容包括：本发明公开了一种人肠道防御素5衍生线性多肽,具有以下通式：SEQ ID NO:1AX<Sub>1</Sub>AX<Sub>2</Sub>ARX<Sub>3</Sub>GRAAX<Sub>4</Sub>RX<Sub>5</Sub>X<Sub>6</Sub>LX<Sub>7</Sub>GVAX<Sub>8</Sub>IX<Sub>9</Sub>GRLYRLAAR。本发明还公开了该多肽的制备方法和应用。本发明的多肽去除了三对定向搭配的二硫键,将剩余非疏水和非正电荷氨基酸替换为精氨酸,简化了多肽合成过程的同时,显著强化了多肽的抗菌活性。(The invention discloses a human intestinal defensin 5 derived linear polypeptide, which has the following general formula: 1AX SEQ ID NO 1 AX 2 ARX 3 GRAAX 4 RX 5 X 6 LX 7 GVAX 8 IX 9 GRLYRLAAR are provided. The invention also discloses a preparation method and application of the polypeptide. The polypeptide removes three pairs of directional collocated disulfide bonds, and replaces the residual non-hydrophobic and non-positive charge amino acids with arginine, thereby simplifying the synthesis process of the polypeptide and simultaneously obviously strengthening the antibacterial activity of the polypeptide.)

1. A human gut defensin 5 derived linear polypeptide having the general formula: 1AX SEQ ID NO₁AX₂ARX₃GRAAX₄RX₅X₆LX₇GVAX₈IX₉GRLYRLAAR，

Wherein, X₁One selected from Thr, Lys or Arg;

X₂one selected from Tyr, Lys or Arg, preferably one from Lys or Arg;

X₃one selected from Thr, Lys or Arg, preferably one selected from Lys or Arg;

X₄one selected from Thr, Lys or Arg, preferably one selected from Lys or Arg;

X₅one selected from Glu, Lys or Arg, preferably one of Lys or Arg;

X₆one selected from Ser, Lys or Arg, preferably one selected from Lys or Arg;

X₇one selected from Ser, Lys or Arg, preferably one selected from Lys or Arg;

X₈one selected from Glu, Lys or Arg, preferably one of Lys or Arg;

X₉is selected from Ser, Lys or Arg, preferably Lys or Arg.

2. The polypeptide of claim 1, wherein the human gut defensin 5 derived linear peptide has the sequence of SEQ ID NO 2ARARARRGRAARRRRLRGVARIRGRL YRLAAR.

3. The polypeptide of claim 1 or 2, wherein the polypeptide has a random coil in aqueous solution and a helical structure in a lipid environment.

4. A method of producing a polypeptide according to any one of claims 1 to 3, comprising:

1) weighing an appropriate amount of 2-Cl (Trt) -Cl resin, adding into a reactor of a polypeptide synthesizer, soaking with Dichloromethane (DCM), and then sequentially washing with Dimethylformamide (DMF) and DCM;

2) adding a proper amount of N' - [ (2, 3-dihydro-2, 2,4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl ] -N-fluorenylmethoxycarbonyl-D-arginine (Fmoc-Arg (pbf) -OH) and 16mL of DCM and 1mL of N, N-Diisopropylethylamine (DIEA) into the polypeptide synthesis reactor in the step 1), and reacting for 90 min; wherein, the material proportion is that, the resin amount: DCM: DIEA 1 g: 14-18 mL: 0.9-1.1mL, preferably resin amount: DCM: DIEA 1 g: 16mL of: 1 mL; and 0.399mmol of N' - [ (2, 3-dihydro-2, 2,4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl ] -N-fluorenylmethoxycarbonyl-D-arginine (Fmoc-Arg (pbf) -OH) was added per 1g of resin.

3) Adding 0.5mL of analytical methanol and 1mL of DCM into the polypeptide synthesis reactor, carrying out blocking reaction for 20min, washing with DMF,

4) adding a piperidine remover to remove 9-fluorenylmethyloxycarbonyl (Fmoc), and washing the resin with DMF (dimethyl formamide); the piperidine removing agent is prepared by mixing piperidine and DMF in a ratio of 1: 4 in a volume ratio;

5) feeding materials in sequence according to the sequence of amino acids in the polypeptide sequence;

6) adding a 1-hydroxybenzotriazole (HoBt)/N, N-Diisopropylcarbodiimide (DIC)/DMF system into a reactor, and adding DMF for reaction;

7) deprotection with piperidine remover until the final Lys deprotection is complete, then washing sequentially with DMF and methanol, and draining;

8) pouring the resin into a 50mL centrifuge tube, adding cutting fluid, and cutting at room temperature for 1.5 h; wherein the cutting fluid is prepared from trifluoroacetic acid (TFA), Triisopropylsilane (TIS), 1, 2-Ethanedithiol (EDT) and water in a volume ratio of 95: 2: 2: 1 in proportion;

9) filtering and taking filtrate, adding glacial ethyl ether into the filtrate for washing to obtain a crude product.

5. The method of claim 4, wherein the amino acids are each weighed in an appropriate amount in step 5) to give Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Tyr-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, and (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH.

6. The method according to claim 4 or 5, wherein in step 9), the volume ratio of the filtrate to the ether is 1: 8, shaking up, centrifuging at 3000rpm for 2min, pouring out supernatant, adding ether, shaking up and centrifuging.

7. The method of any one of claims 4-6, further comprising the following purification steps:

10) the crude product from step 9) is purified by liquid chromatography.

8. The method according to claim 7, wherein in the liquid chromatography of step 10), the column is a daisogel chromatography packing; the mobile phase A is TFA aqueous solution with the volume fraction of 0.1 percent; and the mobile phase B is formed by mixing TFA and acetonitrile, wherein the volume fraction of TFA is 0.1%.

9. An antimicrobial composition comprising the polypeptide of any one of claims 1-3.

10. Use of a polypeptide according to any one of claims 1 to 3 for the preparation of an antibacterial composition.

Technical Field

The application relates to the technical field of biological pharmacy and polypeptide synthesis, in particular to a human intestinal defensin 5 derived linear polypeptide and a preparation method and application thereof.

Background

Acinetobacter baumannii (baumannii) is a conditioned pathogen for nosocomial infection and community acquisition, and is extremely easy to resist drugs. At present, antibiotics for clinically preventing and treating drug-resistant Bowman's infection are in short supply, and part of drugs with obvious toxic and side effects, such as polymyxin, are still used in the first line. In 2017, 2 and 27 months, the world health organization has listed drug-resistant Bowman as the first pathogenic bacterium that urgently needs to develop novel antibiotics.

The antibacterial peptide is a kind of amphiphilic low molecular weight polypeptide widely existing in animal and plant bodies. As an important component molecule of the innate immunity barrier, the antibacterial peptide can effectively protect the organism from being attacked by pathogenic bacteria. Theoretically, pathogenic bacteria do not develop resistance to antimicrobial peptides, and as such, it is hoped that antimicrobial peptides can be developed as a new generation antibiotic (R.E. Hancock, Lancet, 1999,349(3): 418-). 422). Under the background of increasingly severe global bacterial drug resistance situation, more than 10 antibacterial peptide drugs aiming at different bacteria are on the market or enter a clinical test stage (Breij A De. et al, scientific relative Medicine,2018,10(423): eaan4044), but no antibacterial peptide capable of effectively controlling drug-resistant Bowman infection exists in the market.

Human endogenous antibacterial peptide defensins are classified into two main classes α and β according to structural homology and disulfide bond pairing modes, human defensin 5(HD5) is mainly expressed and secreted by intestinal Pan cells and genital tract mucosal epithelial cells and is structurally characterized by ① 32 amino acid residues, wherein 6 basic amino acids (arginine), 2 acidic amino acids (glutamic acid) carry 4 positive charges, ② disulfide bonds are directionally matched in a Cys1-Cys6/Cys2-Cys4/Cys3-Cys5 mode, ③ monomeric peptides present β -folded lamellar-like structures and can be polymerized in a concentration-dependent manner, the antibacterial effect is enhanced, the anti-drug-resistant abalone activity of HD5 is limited in a complex environment, and the drug development of the polypeptide structure is simplified through enhancing and enhancing (Wang, C.30etal, anti-microbial rob Agents Chemothers, 2017, AAC.01504-17) research, and the positive charge effect is determined by Wang, C.30et 84, early stage of the polypeptide structure (Wang, C.30et 83, early stage of the antibiotic effect).

Recombinant expression and chemical synthesis by using genetic engineering technology are two methods for preparing high-purity antibacterial peptide. Compared with a recombinant expression scheme with complex procedures, the chemical synthesis method is simple and direct, and is more beneficial to large-scale industrial preparation of the medicine. HD5 consists of 32L amino acids (SEQ ID NO:3ATCYCRTGRCATRESLSGVCEISGRLYRLCCR) with 3 pairs of aligned paired disulfide bonds. During preparation, in order to prevent disulfide bond mismatching, cysteine sulfydryl is required to be subjected to alkylation blocking in sequence, and a synthetic product is also required to be repeatedly purified. The complex synthesis process greatly improves the preparation cost of the polypeptide and is not beneficial to later development and application. If the disulfide bond of HD5 could be removed, the synthesis of the polypeptide would be significantly simplified. In fact, HD5 formed 3 pairs of complex disulfide bonds, mainly due to the large amount of trypsin contained in the intestinal lumen, while the targeted conjugation of disulfide bonds protected the polypeptide from protease degradation (Wanniarachhi YA. et al, Biochemistry,2011,50: 8005-17). Bowman has a high probability of causing infection in local tissues such as the lung and the skin. Due to the low local tissue trypsin content, the polypeptide does not need to form complex conformation by using disulfide bonds to avoid enzymolysis.

In conclusion, drug-resistant Bowman's infection seriously threatens human health, and the development of novel antibiotics is imminent. The human endogenous antibacterial peptide HD5 has drug-resistant Bowman-resistant activity and is not easily tolerated by bacteria. HD5 is susceptible to interference in a body fluid environment, and has reduced antibacterial effect; the complex disulfide bond pairing of the polypeptide increases the chemical synthesis cost and is not beneficial to industrial preparation and production. Increasing the arginine content of the polypeptide and removing the disulfide bond pairing are expected to advance the medicinal development of HD 5.

Disclosure of Invention

The application aims to provide the multiple drug resistance acinetobacter baumannii polypeptide which is convenient for artificial synthesis and beneficial to industrial preparation and production, and is used for overcoming the defects of shortage of the existing antibiotics and limited treatment effect.

To achieve the above objects, the present application provides a human intestinal defensin 5 derived linear polypeptide having the following general formula:

SEQ ID NO:1AX₁AX₂ARX₃GRAAX₄RX₅X₆LX₇GVAX₈IX₉GRLYRLA

AR，

wherein, X₁One selected from Thr, Lys or Arg;

X₂one selected from Tyr, Lys or Arg, preferably one from Lys or Arg;

X₃one selected from Thr, Lys or Arg, preferably one selected from Lys or Arg;

X₄one selected from Thr, Lys or Arg, preferably one selected from Lys or Arg;

X₅one selected from Glu, Lys or Arg, preferably one of Lys or Arg;

X₆one selected from Ser, Lys or Arg, preferably one selected from Lys or Arg;

X₇one selected from Ser, Lys or Arg, preferably one selected from Lys or Arg;

X₈one selected from Glu, Lys or Arg, preferably one of Lys or Arg;

X₉is selected from Ser, Lys or Arg, preferably Lys or Arg.

In one embodiment according to the invention, the sequence of the human intestinal defensin 5 derived linear peptide is SEQ ID NO. 2ARARARRGRAARRRRLRGVARIRGRL YRLAAR. After the non-basic amino acid of HD5 is mutated into arginine, the antibacterial activity of the polypeptide is obviously improved. Arginine is not only associated with electrostatic adsorption of polypeptides and formation of gaussian curvature, but also facilitates transmembrane localization of molecules and toxin neutralization capacity.

In one embodiment according to the invention, the polypeptide has a random coil in aqueous solution and a helical structure in a lipid environment.

The invention also provides a preparation method of the polypeptide, which comprises the following steps:

1) weighing an appropriate amount of 2-Cl (Trt) -Cl resin, adding into a reactor of a polypeptide synthesizer, soaking with Dichloromethane (DCM), and then sequentially washing with Dimethylformamide (DMF) and DCM;

2) adding 0.399mmol of N' - [ (2, 3-dihydro-2, 2,4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl ] -N-fluorenylmethoxycarbonyl-D-arginine (Fmoc-Arg (pbf) -OH) and DCM and N, N-Diisopropylethylamine (DIEA) into the polypeptide synthesis reactor in the step 1) and reacting for 90 min; the amount of Fmoc-Arg (pbf) -OH used is related to the amount of resin and the degree of substitution; as the peptide chain of the invention belongs to medium-length peptide, the substitution degree is 0.3 mmol/g; the amount of resin used was 1g, and the molar amount of Fmoc-Arg (pbf) -OH was 0.3mmol (1 g. times.0.3 mmol/g); since the condensation efficiency is about 70%, and the weighed amount is 0.399mmol (0.3 × 1.33) in actual synthesis, the material ratio in the preparation method of the present invention is that, the material ratio is that, the resin amount: DCM: DIEA 1 g: 14-18 mL: 0.9-1.1mL, preferably resin amount: DCM: DIEA 1 g: 16mL of: 1 mL; . Wherein 0.399mmol of N' - [ (2, 3-dihydro-2, 2,4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl ] -N-fluorenylmethoxycarbonyl-D-arginine (Fmoc-Arg (pbf) -OH) was added per 1g of resin.

3) Adding appropriate amounts of assay methanol and DCM to the polypeptide synthesis reactor, wherein the volume ratio of methanol to DCM is 1: 2, after the blocking reaction is carried out for 20min, washing with DMF,

4) adding a piperidine removing agent, wherein the piperidine removing agent is prepared by mixing piperidine and DMF at a ratio of 1: 4, removing 9-fluorenylmethyloxycarbonyl (Fmoc), and washing the resin with DMF;

5) feeding materials in sequence according to the sequence of amino acids in the polypeptide sequence; preferably, the amount of the feed is fed in excess, preferably 3 times the actual required molar amount; the 3-fold molar weight is the most economical and effective method for reaction, and more than 3-fold molar weight can increase the cost, less than 3-fold molar weight and prolong the synthesis time.

6) Adding a 1-hydroxybenzotriazole (HoBt)/N, N-Diisopropylcarbodiimide (DIC)/DMF system into a reactor, adding DMF, reacting for 1h, and washing with DMF, preferably 4 times;

7) deprotection with piperidine remover until the final Lys deprotection is complete, washing sequentially with DMF and methanol, and draining;

8) pouring the resin into a 50mL centrifuge tube, adding cutting fluid, and cutting at room temperature for 1.5 h; wherein the cleavage solution is prepared from trifluoroacetic acid (TFA), Triisopropylsilane (TIS), 1, 2-Ethanedithiol (EDT) and water in a ratio of 95: 2: 2: 1 by volume ratio; preferably, the ratio of the mass of the resin to the volume of the cutting fluid is 1g/10 mL; under the condition, the resin peptide has good fluidity in cutting fluid, and amino acid side chain protecting groups can be completely cut off in effective time; the excessive cutting fluid only increases the amount of ether in the subsequent treatment, which causes unnecessary cost waste.

9) Filtering to obtain filtrate, adding glacial ethyl ether into the filtrate, and washing for several times to obtain a crude product.

In one embodiment according to the present invention, in step 5), an appropriate amount of amino acids is weighed in the following order to charge Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Tyr-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Pboc (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ala-OH.

In one embodiment according to the present invention, in step 9), the volume ratio of the filtrate to diethyl ether is 1: 8, shaking up, centrifuging at 3000rpm for 2min, pouring out supernatant, adding ether, shaking up, centrifuging, washing and centrifuging repeatedly for three times.

In one embodiment according to the present invention, the preparation method further comprises the following purification steps:

10) the crude product from step 9) is purified by liquid chromatography.

In one embodiment according to the present invention, in the liquid chromatography in step 10), the column is a daisogel chromatography packing; the mobile phase A is TFA aqueous solution with the volume fraction of 0.1 percent; and the mobile phase B is formed by mixing TFA and acetonitrile, wherein the volume fraction of TFA is 0.1%.

The invention also provides an antibacterial composition comprising a polypeptide according to any one of claims 1-3.

The invention further provides application of the polypeptide in preparing an antibacterial composition.

This application has following advantage:

the polypeptide is rich in arginine with positive charge, does not contain disulfide bond, and the arginine with positive charge is highly conserved in a human α -defensin sequence, the antibacterial activity of the polypeptide is obviously improved after the non-basic amino acid of HD5 is changed into the arginine, and the arginine is not only related to the electrostatic adsorption of the polypeptide and the formation of Gaussian curvature, but also beneficial to the transmembrane positioning of molecules and the toxin neutralization capacity.

The structure elasticity is good. Compared with the parent peptide HD5, the derived linear peptide has the advantages that the anti-multiple drug resistance Bowman activity is obviously enhanced, and the synthesis difficulty and the cost are greatly reduced; compared with the traditional antibiotics, the linear peptide has the advantages that the capacity of treating multiple drug-resistant Bowman lung infected mice is obviously improved, and the medicinal value is good.

Drawings

Fig. 1 is a diagram of the design concept of the AR32 polypeptide of the present application.

Fig. 2 is a liquid chromatogram of an AR32 polypeptide of the present application.

Fig. 3 is a mass spectrometric identification of the AR32 polypeptides of the present application.

Figure 4 is a circular dichroism scan plot of HD5 polypeptide and the AR32 polypeptide of the present application in aqueous solution and Sodium Dodecyl Sulfate (SDS) solution.

Fig. 5 is a bar graph of the results of the biosafety assessment of AR32 polypeptides of the present application.

FIG. 6 is a graph showing the results of an experiment in which an AR32 polypeptide was used to infect animals with radiation wound.

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.