CN107298707B - Bac 5-like antibacterial peptide and application thereof - Google Patents
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Abstract
本发明公开了一种类Bac5抗菌肽，其氨基酸序列如SEQ ID NO.1，其分子量为4480.51Da，等电点为12.6。所述的类Bac5抗菌肽采用自动多肽合成仪按照常规多肽固相法合成。本发明的类Bac5抗菌肽对革兰阳性菌(金黄色葡萄球菌)、革兰阴性菌(大肠杆菌、沙门菌)都有高效的抗菌活性，抗菌作用快，具有低溶血性，不产生耐药性。因此，本发明的类Bac5抗菌肽在制备抗感染药物、畜牧业发展、人类疾病治疗方面具有广阔的应用前景。The invention discloses a Bac5 antibacterial peptide, the amino acid sequence of which is as SEQ ID NO.1, the molecular weight of which is 4480.51 Da, and the isoelectric point is 12.6. The Bac5-like antibacterial peptide is synthesized by an automatic peptide synthesizer according to a conventional peptide solid-phase method. The Bac5-like antibacterial peptide of the present invention has efficient antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Salmonella), has fast antibacterial effect, has low hemolysis, and does not produce drug resistance sex. Therefore, the Bac5-like antimicrobial peptide of the present invention has broad application prospects in the preparation of anti-infective drugs, the development of animal husbandry, and the treatment of human diseases.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to Bac 5-like antibacterial peptide and application thereof.
Background
The discovery of antibiotics has brought enormous benefits to humans. However, with the heavy use and abuse of antibiotics, bacterial drug resistance is continuously generated, veterinary drug residue harms are continuously generated, and the human health and the animal husbandry development are seriously harmed. The search for new antibacterial agents is becoming more urgent. In the search, people gradually focused their eyes on antimicrobial peptides. Antimicrobial peptides (AMPs) are important components of the innate immune system of a biological organism, are composed of 10-60 amino acid residues, have biological activities such as antibacterial activity, antiviral activity, antiparasitic activity, antitumor activity, immunoregulation activity and the like, are paid attention to due to the characteristics of small molecular weight, good thermal stability, good water solubility, wide Antimicrobial spectrum, difficulty in forming drug resistance and the like, and are considered to be the best substitute of antibiotics.
At present, research and application of antibacterial peptides have become hot spots in the field of biotechnology, and many antibacterial peptides have been developed into clinical drugs. The antibacterial peptide Bac5 was isolated from bovine neutrophils in 1989, is an antibacterial peptide consisting of 43 amino acids and rich in arginine and proline, and has broad-spectrum antibacterial activity. However, most of natural antibacterial peptides have low bacteriostatic activity, and often have toxicity and hemolytic effect on normal eukaryotic cells while exerting bacteriostatic effect, and the low yield of natural antibacterial peptides limits the development and application of antibacterial peptides. According to the structure-activity relationship and the action mechanism of the antibacterial peptide, the artificial modification and design of the existing antibacterial peptide by taking the excellent antibacterial peptide as a template are an important direction for developing the antibacterial peptide. Compared with natural antibacterial peptide, the artificially designed antibacterial peptide has better target cell specificity and antibacterial activity, and even has the characteristics of low hemolysis, low sensitivity to protease degradation and the like. Therefore, Bac5 is used for designing Bac 5-like antibacterial peptide which is simple in structure, easy to chemically synthesize and high in antibacterial activity.
Disclosure of Invention
The invention aims to provide Bac 5-like antibacterial peptide and application thereof. The Bac 5-like antibacterial peptide disclosed by the invention has high-efficiency antibacterial activity on gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli and salmonella), is quick in antibacterial action, has low hemolytic property, and does not generate drug resistance. Therefore, the Bac 5-like antibacterial peptide has wide application prospects in the aspects of preparing anti-infective medicaments, developing animal husbandry and treating human diseases.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
bac 5-like antibacterial peptide, which has the following amino acid sequence:
phenylalanine-arginine-proline-isoleucine-arginine-proline-phenylalanine-tyrosine-proline-phenylalanine-arginine-proline-isoleucine-phenylalanine-proline-isoleucine-arginine-proline-phenylalanine-arginine-proline (SEQ ID No. 1).
According to the Bac 5-like antibacterial peptide, the Bac 5-like antibacterial peptide has 37 amino acid residues, the molecular weight of the Bac 5-like antibacterial peptide is 4480.51Da, and the isoelectric point of the Bac 5-like antibacterial peptide is 12.6.
The Bac 5-like antibacterial peptide can be used for preparing medicaments for treating gram-positive bacteria and gram-negative bacteria infection. The medicament comprises Bac 5-like antibacterial peptide and one or more than one pharmaceutically acceptable carriers and/or additives.
The Bac 5-like antibacterial peptide can also be used for preparing feed additives, disinfectants, preservatives or cosmetic additives.
The Bac 5-like antibacterial peptide is synthesized by an automatic polypeptide synthesizer according to a conventional polypeptide solid phase method, the synthesis direction is from a C end to an N end, and the synthesis comprises the following detailed steps:
a. swelling resin: adding Fmoc-Pro-Wang resin into a reactor of an automatic polypeptide synthesizer, adding dimethyl formamide DMF (dimethyl formamide) for swelling, wherein the amount of the 1g of Fmoc-Leu-Wang resin added into the DMF is about 12mL (the DMF completely immerses the resin), the swelling time is 10-30 min, and the swelling can be repeated for 1-2 times;
b. deprotection: after the resin is swelled, 20% of piperidine (20% of piperidine is prepared by dissolving piperidine in dimethylformamide DMF) in a storage tank is added into a reactor, the resin is immersed (about 10mL of 20% of piperidine is required to be added into 1g of Fmoc-Pro-Wangerinin resin), the swelled resin is deprotected for about 30min, and then dimethylformamide DMF is added for washing;
c. condensation reaction: then adding a second amino acid proline Fmoc-Pro according to the sequence of Bac 5-like antibacterial peptide amino acid sequence, and adding a condensing agent HCTU (6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea hexafluorophosphate) and a catalyst NMM (N-methylmorpholine) or DIEA (N, N diisopropylethylamine) for condensation reaction; after the reaction is finished, adding dimethyl formamide DMF for repeated washing to remove unreacted amino acid; the molar ratio of the proline to the Fmoc-Pro-Wang resin is 5: 1; the molar ratio of the condensing agent HCTU to the proline Fmoc-Pro is 1: 1; the molar ratio of the catalyst NMM or DIEA to proline is 4: 1;
deprotecting and washing the product obtained after washing in the same manner as in step b, and condensing Fmoc-Arg, Fmoc-Phe, Fmoc-Pro, Fmoc-Arg, Fmoc-Ile, Fmoc-Pro, Fmoc-Phe, Fmoc-Ile, Fmoc-Pro, Fmoc-Arg, Fmoc-Phe, Fmoc-Pro, Fmoc-Tyr, Fmoc-Phe, Fmoc-Pro, Fmoc-Arg, Fmoc-Ile, Fmoc-Pro, Fmoc-Arg, Fmoc-Ile, Fmoc-Pro, Fmoc-Arg, Fmoc-Phe, Fmoc-Arg, and Fmoc-Phe, Washing, and obtaining a polypeptide product after the last condensation reaction and washing; carrying out deprotection and washing on products obtained after each condensation reaction and washing according to the method in the step b, and then adding the next amino acid to carry out condensation reaction and washing (in each condensation reaction, the molar ratio of the added amino acid to Fmoc-Pro-Wang resin is 5: 1; the molar ratio of the condensing agent HCTU to each added amino acid is 1: 1; and the molar ratio of the catalyst NMM or DIEA to each added amino acid is 4: 1);
d. deprotection and cleavage of the polypeptide product: adding 20% piperidine to the polypeptide product from step c and submerging the polypeptide product (about 10mL of 20% piperidine was used to deprotect 1g of the resin) to deprotect the polypeptide product; after deprotection, sequentially adding dimethylformamide DMF and dichloromethane DCM for repeated washing; after washing, the polypeptide is cleaved from the resin by addition of trifluoroacetic acid, TFA (1g of resin is cleaved with TFA in a volume of about 20 mL); filtering the cracked product (by using a sand core funnel) to obtain a filtrate;
e. and (3) precipitating and synthesizing the polypeptide: washing the obtained filtrate with cold ether, centrifuging to precipitate and synthesize polypeptide, wherein the process comprises the following steps: d, adding cold diethyl ether (preserved at 4 ℃ in advance) with the volume 3 times of that of the filtrate obtained in the step d, sealing, reversing, uniformly mixing, carrying out ice bath for 10min, centrifuging at 4000r/m for 10min, removing supernatant, and recovering precipitate; re-suspending the precipitate with cold ether, repeating the steps for three times, and drying the obtained precipitate for 12-16 h to obtain a crude synthetic peptide product;
f. purification of crude synthetic peptide: and (3) performing column purification on the synthetic peptide crude product by adopting a semi-preparative high performance liquid chromatograph P2000, and collecting fractions with the purity of more than or equal to 95% to obtain the purified Bac 5-like antibacterial peptide with the purity of more than or equal to 95%.
A semi-preparative high performance liquid chromatograph P2000 is provided by Beijing Wawawa Innovation technologies, Inc., a C18 reverse phase column (4.6 x 250mm) is adopted as a chromatographic column, 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution) is adopted as a mobile phase A, 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water) is adopted as a mobile phase B, the proportion of the mobile phase A is gradually adjusted to 52% from 27% within 25min after sample loading by using the processing column, the proportion of the mobile phase B is gradually adjusted to 48% from 73%, the proportion of the mobile phase A is changed to 100% and the proportion of the mobile phase B is changed to 0% during 25.1min, separation is carried out for 30min at the flow rate of 1mL/min, the detection wavelength is 220nm, and fractions with the purity of more than or equal to 95% are collected (as shown in figure 1);
the Bac 5-like antibacterial peptide obtained by the invention is confirmed and identified as follows: the molecular weight of the antibacterial peptide of the obtained product is determined by a liquid chromatography-mass spectrometer (Watersicromass ZQ-2000), and the determination is as follows: it has a molecular weight of 4480.51Da (as shown in FIG. 2). High performance liquid chromatography conditions: the chromatographic column is a C18 reverse phase column (4.6 × 250mm), the mobile phase A is 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution), the mobile phase B is 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water), the proportion of the mobile phase A is gradually adjusted from 25% to 50% within 25min after loading, the proportion of the mobile phase B is gradually adjusted from 75% to 50%, the proportion of the mobile phase A is changed to 100% and the proportion of the mobile phase B is changed to 0% within 25.1min, separation is carried out at the flow rate of 1mL/min for 30min, and the detection wavelength is 220 nm. Mass spectrometry conditions: and adopting a positive ionization mode, wherein the capillary voltage is 3.00KV, the outlet voltage of the capillary is 50V, the fragmentation voltage is 5V, the flow rate of the drying gas is 1.5L/min, the temperature of the drying gas is 350 ℃, and the scanning range is 400-1900 m/z.
The invention has the following positive beneficial effects:
the Bac 5-like antibacterial peptide consists of 37 amino acids, is rich in arginine, has high-efficiency antibacterial activity on gram-positive bacteria (staphylococcus aureus), gram-negative bacteria (escherichia coli and salmonella) and fungi, has quick antibacterial action and strong antibacterial activity, has low hemolysis and does not generate drug resistance. Therefore, the Bac 5-like antibacterial peptide has wide application prospects in the aspects of preparing anti-infective medicaments, developing animal husbandry and treating human diseases.
Drawings
FIG. 1 is a high performance liquid chromatogram of Bac 5-like antibacterial peptide.
FIG. 2 is a mass spectrum of Bac 5-like antibacterial peptide.
FIG. 3 shows the results of isoelectric point analysis of Bac 5-like antibacterial peptide.
FIG. 4 shows the result of the minimum inhibitory concentration of Bac 5-like antibacterial peptide on Escherichia coli.
FIG. 5 shows the result of the Bac 5-like antibacterial peptide on the determination of the minimum inhibitory concentration of the salmonella.
FIG. 6 shows the result of the minimum inhibitory concentration of Bac 5-like antibacterial peptide on Staphylococcus aureus.
FIG. 7 shows the result of measurement of the hemolysis rate of Bac 5-like antibacterial peptide.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
Example 1:
bac 5-like antibacterial peptide, which has the following amino acid sequence:
phenylalanine-arginine-proline-isoleucine-arginine-proline-phenylalanine-tyrosine-proline-phenylalanine-arginine-proline-isoleucine-phenylalanine-proline-isoleucine-arginine-proline.
The Bac5 antibacterial peptide contains 37 amino acid residues, the molecular weight of the Bac5 antibacterial peptide is 4480.51Da, and the isoelectric point of the Bac5 antibacterial peptide is 12.6. The Bac 5-like antibacterial peptide is rich in arginine residues (positively charged) and can attract negative charges on the surface of bacteria.
The Bac5 antibacterial peptide is synthesized by an automatic polypeptide synthesizer according to a conventional polypeptide solid phase method, and finally the synthesized Bac5 antibacterial peptide is purified and analyzed by a semi-preparative high performance liquid chromatograph, wherein the purity of the Bac5 antibacterial peptide is more than or equal to 95%.
The Bac 5-like antibacterial peptide product obtained by the invention is identified as follows:
(1) determination of molecular weight: the molecular weight of the Bac 5-like antibacterial peptide is determined by an analytical liquid chromatography-mass spectrometer (Waters micromass ZQ-2000), and the molecular weight is 4480.51Da, and the result is shown in figure 2.
The specific detection conditions are ① chromatographic conditions, namely a C18 reverse phase column (4.6 mm by 250mm), a 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution), a 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water) is adopted as a mobile phase A, the proportion of the mobile phase A is gradually adjusted from 25% to 50% within 25min after sample loading, the proportion of the mobile phase B is gradually adjusted from 75% to 50%, the proportion of the mobile phase A is changed to 100% within 25min after sample loading, the proportion of the mobile phase B is changed to 0%, separation is carried out at the flow rate of 1mL/min for 30min, the detection wavelength is 220nm, ② mass spectrometry is carried out under the conditions that a normal mode is adopted, the capillary voltage is 3.00KV, the capillary outlet voltage is 50V, the fragmentation voltage is 5V, the air flow rate is dried to 1.5L/min, the drying air temperature is 350 ℃, and the scanning range is 400-1900/z.
(2) Amino acid sequence structure determination: and (3) determining the amino acid sequence structure of the Bac 5-like antibacterial peptide by adopting an automatic amino acid sequencer. Through determination, Bac 5-like antibacterial peptide contains 37 amino acid residues, and the amino acid sequence of the Bac 5-like antibacterial peptide is as follows: phenylalanine-arginine-proline-isoleucine-arginine-proline-phenylalanine-tyrosine-proline-phenylalanine-arginine-proline-isoleucine-phenylalanine-proline-isoleucine-arginine-proline.
(3) Isoelectric point analysis:
opening EditSeq in DNAStar software, opening file, clicking new protein in new, inputting an amino acid sequence of Bac 5-like antibacterial peptide, and storing the amino acid sequence as a pro-format document. And then opening a Protean in DNAStar software, opening a file, clicking "open", selecting the document just stored in the pro format, clicking "open", and selecting "transformation cut" in "Analysis", so that the isoelectric point data can be obtained, wherein the result is shown in figure 3. Through determination, the isoelectric points of Bac 5-like antibacterial peptides are respectively 12.6.
Example 2: application implementation of Bac 5-like antibacterial peptide
1. Antibacterial activity analysis of Bac 5-like antibacterial peptide
The antibacterial activity of Bac 5-like antibacterial peptide is measured by adopting an agar plate diffusion method, and tested microbial strains are as follows: escherichia coli ATCC8099, salmonella CVCC541 and staphylococcus aureus ATCC6538, wherein the Escherichia coli and the staphylococcus aureus are purchased from Nanjing fecal diagnosis Biotech limited company, and the salmonella is purchased from China veterinary microorganism culture collection management center.
The 3 test microorganisms were diluted in 50 ℃ basal medium (TSB 0.3g, ultrapure agarose 0.1g, ultrapure water to 10ml, pH 7.4) respectively, the plates were poured out and sterilized, coagulated, punched with a punch (pore diameter: about 3mm), bottom-sealed with alcohol lamp by slight heating, and 5. mu.L of antimicrobial peptide sample was added to each well with a pipette, each plate including positive and negative controls. Positive control: ampicillin is directed against gram-negative and gram-positive bacteria; negative control: PBS buffer. The plate was allowed to stand for 1h to allow the test solution to diffuse into the agarose. Then, an upper layer of overlay medium (TSB 0.3g, ultrapure agarose 0.1g, ultrapure water to 10ml, pH 7.4, 50 ℃ C. or so) was added thereto. The plates were incubated at 37 ℃ overnight in an inverted manner, and then the diameter of the transparent circle around the well was recorded, and the measurement was repeated three times for each strain, and the average value was calculated.
TABLE 1 results of antibacterial activity analysis of Bac 5-like antibacterial peptides
The antibacterial activity detection results in table 1 show that the Bac 5-like antibacterial peptide has stronger antibacterial activity on escherichia coli, salmonella and staphylococcus aureus. Therefore, the Bac 5-like antibacterial peptide of the invention is expected to have good application prospect in the aspect of preparing drugs for treating animal and human bacterial diseases, and meanwhile, according to the general characteristics of the known antibacterial peptide, the Bac 5-like antibacterial peptide has good application potential in the aspects of feed additives, disinfectants, preservatives, cosmetic additives and the like.
2. Minimum Inhibitory Concentration (MIC) determination of Bac 5-like antibacterial peptide
The tested microbial strains were: escherichia coli ATCC8099, salmonella CVCC541 and staphylococcus aureus ATCC6538, wherein the Escherichia coli and the staphylococcus aureus are purchased from Nanjing fecal diagnosis Biotech limited company, and the salmonella is purchased from China veterinary microorganism culture collection management center.
The tested strain is enlarged and cultured in TSB liquid culture medium, and OD of the bacterial liquid is measured600The value is obtained. When OD value is 0.6-0.8, centrifuging the bacterial solution at 6000r/min for 10min, discarding supernatant, collecting thallus precipitate, re-suspending the thallus precipitate with PBS buffer solution (0.01M) of the same volume, and re-suspending with MH Medium dilution of bacterial suspension to 2X 106CFU/mL. And diluting the Bac 5-like antibacterial peptide sample with deionized water to ensure that the concentration of the Bac 5-like antibacterial peptide sample is 0.78-100 mu g/mL. Respectively adding 50 μ L Bac 5-like antibacterial peptide with different concentrations and 50 μ L diluted bacteria solution into each well of 96-well cell culture plate, repeating three wells for each concentration, culturing at 37 deg.C for 16-18h, mixing the solutions in each well, and determining OD600Values were given as their MIC in Bac 5-like antimicrobial peptide concentrations corresponding to sudden changes in OD values, and the results are shown in fig. 4, 5, and 6.
As can be seen from FIGS. 4, 5 and 6, Bac 5-like antibacterial peptide had a Minimum Inhibitory Concentration (MIC) of 6.25. mu.g/mL for Escherichia coli, 6.25. mu.g/mL for Salmonella and 12.5. mu.g/mL for Staphylococcus aureus. The result shows that the Bac5 antibacterial peptide has the minimum inhibitory concentration reaching microgram level to common bacteria and has extremely strong inhibitory activity.
3. Hemolytic analysis of Bac 5-like antibacterial peptide
Fresh rabbit blood is taken, anticoagulated by 3.8% sodium citrate, the anticoagulated blood is centrifuged for 10min at 3000r/min, and is washed and precipitated for 3 times by Phosphate Buffer Solution (PBS) until the supernatant is colorless and transparent, and then 1% erythrocyte is prepared by PBS. Bac 5-like antibacterial peptide was adjusted to a concentration of 400. mu.g/mL, 200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, 1.56. mu.g/mL, 0.78. mu.g/mL, 0.39. mu.g/mL with PBS, and an equal volume of the red blood cell suspension was added. PBS is used as a negative control, 1% Tritonx-100 is used as a positive control, the reaction is carried out for 1h at 37 ℃, centrifugation is carried out for 10min at 1500r/min, supernatant is sequentially added into a 96-well plate, and the OD value at 540nm is measured by a microplate reader. Then, the hemolysis rate (%) was calculated according to the calculation formula (detection well OD value-negative well OD value)/(positive well OD value-negative well OD value) × 100%, and the results thereof are shown in fig. 7.
As can be seen from FIG. 7, under the high concentration condition of 400. mu.g/mL, the hemolysis rate of Bac 5-like antibacterial peptide is 4.29%, and is still lower than 5%, which indicates that Bac 5-like antibacterial peptide has better safety and huge clinical application prospect.
In conclusion, the Bac 5-like antibacterial peptide product does not produce hemolytic property, has a wide antibacterial spectrum, and has a high-efficiency antibacterial effect on gram-negative bacteria and gram-positive bacteria. Therefore, the Bac5 antibacterial peptide can be preferably applied to preparation of medicaments for treating gram-positive bacteria and gram-negative bacteria, and can also be used for preparing feed additives, disinfectants, preservatives or cosmetic additives.
SEQUENCE LISTING
<110> institute of science and technology of Henan
<120> Bac 5-like antibacterial peptide and application thereof
<130>1
<160>1
<170>PatentIn version 3.5
<210>1
<211>37
<212>PRT
<213>Artificial Sequence
<220>
<223>1
<400>1
Phe Arg Pro Pro Ile Arg Arg Pro Pro Ile Arg Pro Pro Phe Tyr Pro
1 5 10 15
Pro Phe Arg Pro Pro Ile Arg Pro Pro Ile Phe Pro Pro Ile Arg Pro
20 25 30
Pro Phe Arg Pro Pro
35
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| Amino Acid Sequences of Two Proline-rich Bactenecins;Rainer W.Frank;《The journal of biological chemistry》;19900423;第265卷(第31期);18871-18874 * |
| 牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达，纯化及抑菌活性;赵昆等;《中国生物工程杂志》;20070311;第27卷(第3期);65-70 * |
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