CN110317253B - Chicken-derived antibacterial peptide and preparation method thereof - Google Patents
Chicken-derived antibacterial peptide and preparation method thereof Download PDFInfo
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- CN110317253B CN110317253B CN201910676693.9A CN201910676693A CN110317253B CN 110317253 B CN110317253 B CN 110317253B CN 201910676693 A CN201910676693 A CN 201910676693A CN 110317253 B CN110317253 B CN 110317253B
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- A61P31/04—Antibacterial agents
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Abstract
The invention relates to a chicken-derived antibacterial peptide. The sequence of the antibacterial peptide is as follows: <xnotran> MHCLKIMAFLLFFSLLLSQVYCASLHQPQP LLRLKRMTPFWRGVSLRPVGASCRKNSKCITMLCRKNRCFLRTASE, SEQ ID NO:1. </xnotran> The improved chicken antibacterial peptide LEAP-2 contains 76 amino acid residues, has the molecular weight of 8847.8Da, the isoelectric point of 10.61, the positive charge of 13, the instability coefficient of 48.95 and the total average hydrophobicity of 0.195. The preparation method of the improved chicken-derived antibacterial peptide LEAP-2 comprises the following steps: constructing a prokaryotic expression vector pET28a, transforming BL21, inducing expression and purifying protein. The chicken-derived antibacterial peptide can be more efficiently expressed, has broad-spectrum antibacterial property, and compared with the original chicken-derived antibacterial peptide LEAP-2, the improved chicken-derived antibacterial peptide LEAP-2 has high antibacterial activity which is remarkably abnormal and stable and is not easy to inactivate. The chicken-derived antibacterial peptide has the instability coefficient of 48.95, and compared with the original chicken-derived antibacterial peptide, the stability is improved by 11.4 percent.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an improved chicken-derived antimicrobial peptide LEAP-2.
Background
The abuse of antibiotics causes the drug-resistant strains to flood and cause disasters, directly or indirectly harms the physical health of human beings, destroys the living environment of human beings, increases the difficulty of preventing and treating animal diseases, and is one of the most serious problems harming the development of modern breeding industry and the safety of food and medicine. Since the use of antibiotic growth promoters is prohibited in the European Union in 2006, various countries follow up one after another, and China also prohibits the use of antibiotics in the aquaculture industry comprehensively in 2020, so that the search for alternatives for antibiotics is a necessary choice. Among them, antibacterial peptides are considered as the best choice for antibiotic alternatives.
The antibacterial peptide has micromolecule polypeptide with characteristics of strong basicity, thermal stability, broad-spectrum antibiosis and the like, and is widely present in animals, plants and microorganisms. The antibacterial peptide can compete with the cation bound by LPS on the membrane of gram-negative bacteria, and the binding capacity of the antibacterial peptide and LPS is stronger than that of Ca2+ and Mg2 +. Gram-positive bacteria, while lacking LPS, have an abundance of negatively charged teichoic acid and glycoaldehyde teichoic acid in their cell envelope. The amino acid residue with positive charge is firmly adsorbed together after contacting with the outer membrane with negative charge, then the hydrophobic part in the antibacterial peptide protrudes into the cell membrane of the microorganism, the arrangement sequence of the protein originally embedded in the biological membrane on the cell membrane becomes disordered, the membrane potential is depolarized, simultaneously a plurality of antibacterial peptides can be condensed to form a channel penetrating through the whole cell membrane, and the channel can diffuse the ion quantity in the cell out of the cell and then change the osmotic pressure to die.
Staphylococcus aureus (Staphylococcus aureus in Chickens) is an infectious disease caused by staphylococcus, generally regarded as a main pathogenic bacterium, and has various types, which causes great loss to the chicken industry. Clinical manifestations include acute sepsis, arthritis, chicken cord inflammation, skin (including wingtip) necrosis and periostitis. After infection, most chicks are affected by acute septicemia and pathological changes, while the middle chicks are acute or chronic, and the adult chicks are chronic. The death rate of chicks and middle chicks is high, and the disease is one of serious diseases in the poultry industry. Staphylococci have attracted extensive attention in human and veterinary medicine for the last 20 years. On one hand, besides causing a large amount of inflammation of people, enterotoxin can be generated to pollute food, and food poisoning can occur under certain conditions. On the other hand, due to the widespread use of modern antibiotic therapy, the addition of antibiotics to foods (including animal feeds) has resulted in the disease of staphylococci, which originally had only a facultative pathogenic effect, in humans and animals. Therefore, staphylococcus is now one of pathogenic bacteria widely distributed in the world, and attracts general attention.
LEAP-2 (lever expressed antimicrobial peptide-2, LEAP-2) extracted from chicken is LEAP-2 family antimicrobial peptide found in birds at first, and is composed of 76 amino acid residues, a disulfide bond at the C-terminal, homology with human LEAP-2 sequence, 3 exons and 2 introns on chicken chromosome 13. The antimicrobial peptide LEAP-2 is a natural antibiotic in the nature, has a wide range of bacteria species capable of killing, has the functions of inhibiting fungi and causing certain tumor cells and viruses, has the effects of damaging and inhibiting the functions of bacillus subtilis, staphylococcus aureus, escherichia coli and aspergillus flavus, has no toxicity to most animal cells, and has good research prospect and significance. However, the expression quantity of the antimicrobial peptide LEAP-2 in the chicken body is very small, the antimicrobial peptide LEAP-2 is directly extracted from the chicken body in practical application, the cost is higher, the bacteriostatic action is not obvious enough, and the antimicrobial peptide LEAP-2 is difficult to be applied in practical production.
Disclosure of Invention
The invention aims to provide a chicken-derived antibacterial peptide with high antibacterial activity and excellent stability.
The purpose of the invention is realized according to the following technical scheme:
the chicken-derived antibacterial peptide is characterized in that the amino acid sequence is as follows:
MHCLKIMAFLLFFSLLLSQVYCASLHQPQPLLRLKRMTPFWRGVSLRPVGASCRKNSKCITMLCRKNRCFLRTASE，SEQ ID NO:1。
the chicken-derived antibacterial peptide is improved in the chicken-derived antibacterial peptide LEAP-2, and comprises 76 amino acid residues, wherein the molecular weight of the chicken-derived antibacterial peptide is 8847.8Da, the isoelectric point of the chicken-derived antibacterial peptide is 10.61, the positive charge of the chicken-derived antibacterial peptide is 13, the instability coefficient of the chicken-derived antibacterial peptide is 48.95, and the total average hydrophobicity of the chicken-derived antibacterial peptide is 0.195.
The chicken-derived antibacterial peptide is prepared by a biotechnological method known to a person skilled in the art, and the method comprises the following steps: constructing a prokaryotic expression vector pET28a, transforming BL21, inducing expression and purifying protein.
The invention has the following beneficial effects:
the chicken-derived antibacterial peptide can be more efficiently expressed, has broad-spectrum antibacterial property, and compared with the original chicken-derived antibacterial peptide LEAP-2, the improved chicken-derived antibacterial peptide LEAP-2 has high antibacterial activity which is remarkably abnormal and stable and is not easy to inactivate. The chicken-derived antibacterial peptide has the instability coefficient of 48.95, and compared with the original chicken-derived antibacterial peptide, the stability is improved by 11.4 percent.
Drawings
FIG. 1 is a diagram showing a comparison of tertiary structures of the chicken-derived antibacterial peptide of the present invention and the original chicken-derived antibacterial peptide; a is a structure diagram of an original chicken-derived antibacterial peptide LEAP-2, and b is a structure diagram of the chicken-derived antibacterial peptide. As can be seen from FIG. 1, the chicken-derived antimicrobial peptide of the present invention is significantly changed from the original chicken-derived antimicrobial peptide LEAP-2, and the beta-sheet structure at the rear end is changed into an alpha-helix structure.
FIG. 2 measurement of the antibacterial activity of original chicken-derived antibacterial peptide and modified chicken-derived antibacterial peptide LEAP-2 of the present invention;
a: comparing the inhibition rate of the Escherichia coli;
b: comparing the inhibition rate of staphylococcus aureus;
c: comparing the inhibition rate of the bacillus subtilis;
d: comparing the inhibition rate of pseudomonas aeruginosa;
O-LEAP-2 represents the antimicrobial peptide LEAP-2 isolated from the liver cells of chickens;
P-LEAP-2 represents chicken-derived antimicrobial peptide LEAP-2 expressed by Escherichia coli;
LEAP-2-11 shows the improved chicken-derived antibacterial peptide LEAP-2 of the present invention expressed by E.coli.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described below in conjunction with the drawings in the embodiments of the present invention, and the described embodiments are some, but not all embodiments of the present invention.
Example 1 prokaryotic expression and purification of antimicrobial peptides
(1) Construction of prokaryotic expression vector
The prokaryotic expression vector pET28a of the chicken-derived antibacterial peptide is constructed and synthesized by Suzhou Jinweizhi biotechnology and science and technology Limited. The gene sequence of the improved chicken-derived antimicrobial peptide LEAP-2 is as follows:
atgcactgtttgaaaattatggcattccttttattcttctcgctgctgctcagccaggtgtactgtgcttccctgcaccaaccacagccccttctgagactgaagcggatgacgcctttctggagaggagtctcactgagacctgttggagcctcatgtaggaaaaacagtaaatgcatcacaatgctatgcaggaagaaccgctgcttccttagaacggcctccgagtga，SEQ ID NO:2。
(2) Screening of BL21 transformant and induction of xylanase expression
The Escherichia coli DH5 alpha strain containing pET28a-LEAP-2 with correct sequencing is inoculated into LB liquid culture medium, the flask is shaken overnight to extract plasmids, 10 mu L of plasmids are taken to transform 50 mu L of competence BL21, and the transformation method refers to the simple, convenient, fast and efficient Escherichia coli transformation method of Yuyanlin and the like. The transformation system is coated on an LB solid plate containing 50 mu g/mL kanamycin, and after overnight, a single colony is picked for bacteria liquid PCR detection. And inoculating the transformant which is verified to be positive by the PCR of the bacterial liquid into an LB liquid culture medium containing 50 mu g/mL kanamycin resistance, shaking the bacteria until the wavelength is 600nm, adding IPTG (isopropyl thiogalactoside) with the final concentration of 100ug/mL for protein expression induction when (OD) 600=0.7, continuing shaking the bottle for 8 hours at 150r/min, and collecting the thalli.
(3) Purification of proteins
Inoculating the positive transformant into an LB culture medium, shaking the flask at 37 ℃ and 150r/min for culture, taking 200 mu L of bacterial liquid for about 7-8h, putting the bacterial liquid into an enzyme label strip, adding IPTG (isopropyl thiogalactoside) with the final concentration of 100 mu g/mL when passing through an ultraviolet spectrophotometer (OD) 600=0.7 for protein expression induction, continuing to shake the flask at 37 ℃ and 150r/min for 8h, and collecting thalli. The cells were collected by a centrifuge and stored in a refrigerator at-20 ℃.
Taking a proper amount of collected thalli, carrying out ultrasonic disruption on the thalli under the protection of ice after using 50mM phosphate buffer solution to resuspend the thalli, carrying out 7000r/min centrifugation on the disrupted thalli through a centrifuge, carrying out 10min centrifugation, carrying out affinity chromatography separation on the thalli after collecting supernatant, wherein eluents are imidazole solutions with different concentrations (the imidazole solution is proportioned by the phosphate buffer solution), and the imidazole eluents are diluted according to concentration gradients: the method comprises the steps of filtering all liquid passing through a nickel column through a 0.22 mu m filter membrane to be used, collecting proteins eluted by imidazole solutions with different concentrations, performing SDS-PAGE electrophoresis, observing a target strip, dialyzing a mixed solution containing a target protein and an imidazole eluent by using a phosphate buffer solution as a dialyzate and using a semipermeable membrane to remove imidazole, freeze-drying the target protein by using a freeze dryer, subpackaging at-80 ℃, obtaining the target protein with higher concentration, and determining the concentration of the protein (the BCA method protein quantification kit determination).
Example 2 determination of bacteriostatic Activity of modified Chicken-derived antimicrobial peptide LEAP-2
The antibacterial activity of the improved chicken-derived antibacterial peptide LEAP-2 is determined and compared with the original chicken-derived antibacterial peptide LEAP-2. The inhibition rate of the antibacterial peptide on each indicator bacterium is measured when the mass concentration of the antibacterial peptide is respectively 30, 60, 80 and 100 mu g/mL, and the antibacterial activity is further evaluated. Selecting indicator bacteria in 1mL LB culture medium, culturing to logarithmic phase, diluting the bacteria to 1 × 10 with LB culture medium 5 mL -1 Left and right for standby. Dissolving the antibacterial peptide to the required concentration by using sterile 50mmol/L phosphate buffer solution (pH7.0), adding 20 mu L of each concentration of the antibacterial peptide into a 96-well plate, adding 100 mu L of diluted bacterial liquid into each well, paralleling 3 samples, oscillating at 37 ℃ overnight for 16h, and measuring the absorbance of the samples at 600nm by using a microplate reader. Experimental group A 600 The value is A, and the absorbance of a mixture of 20. Mu.L of sterile 50mmol/L phosphate buffer (pH 7.0) and 100. Mu.L of fresh LB medium is A 1 Absorbance of a mixture of 20. Mu.L of sterile 50mmol/L phosphate buffer solution (pH 7.0) and 100. Mu.L of test bacteria was A 0 The inhibition rate was calculated according to the formula (1).
The results are shown in FIG. 2, from which it can be seen that: the improved chicken-derived antibacterial peptide LEAP-2 has broad-spectrum antibacterial activity, and compared with the original chicken-derived antibacterial peptide LEAP-2, the improved chicken-derived antibacterial peptide LEAP-2 has obviously improved antibacterial rate on staphylococcus aureus, bacillus subtilis and pseudomonas aeruginosa. Under the concentration of 100 mu g/mL, the inhibition rates of the improved chicken-derived antibacterial peptide LEAP-2 on staphylococcus aureus, bacillus subtilis and pseudomonas aeruginosa respectively reach 91%,58% and 45%.
Sequence listing
<110> Jilin university of agriculture
<120> an avian antibacterial peptide and its preparation method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 76
<212> PRT
<213> Artificial sequence ()
<400> 1
Met His Cys Leu Lys Ile Met Ala Phe Leu Leu Phe Phe Ser Leu Leu
1 5 10 15
Leu Ser Gln Val Tyr Cys Ala Ser Leu His Gln Pro Gln Pro Leu Leu
20 25 30
Arg Leu Lys Arg Met Thr Pro Phe Trp Arg Gly Val Ser Leu Arg Pro
35 40 45
Val Gly Ala Ser Cys Arg Lys Asn Ser Lys Cys Ile Thr Met Leu Cys
50 55 60
Arg Lys Asn Arg Cys Phe Leu Arg Thr Ala Ser Glu
65 70 75
<210> 2
<211> 231
<212> DNA
<213> Artificial sequence ()
<400> 2
atgcactgtt tgaaaattat ggcattcctt ttattcttct cgctgctgct cagccaggtg 60
tactgtgctt ccctgcacca accacagccc cttctgagac tgaagcggat gacgcctttc 120
tggagaggag tctcactgag acctgttgga gcctcatgta ggaaaaacag taaatgcatc 180
acaatgctat gcaggaagaa ccgctgcttc cttagaacgg cctccgagtg a 231
Claims (2)
1. The chicken-derived antibacterial peptide is characterized by comprising the following antibacterial peptide sequence: MHCLKIMFLFFSLLLSQVYCASLHQPQPLLRLKRMTPFWRGVGSLCRKNSKCITMLCRCKNRCFLRTASE (SEQ ID NO: 1).
2. A method for preparing the chicken-derived antibacterial peptide according to claim 1, comprising the steps of: constructing a prokaryotic expression vector pET28a, transforming BL21, inducing expression and purifying protein.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105483247A (en) * | 2015-12-29 | 2016-04-13 | 云南农业大学 | Method for judging disease resistance of Yunnan native chicken based on LEAP-2 (liver expressed antimicrobial peptide 2) gene |
| CN108314722A (en) * | 2018-04-27 | 2018-07-24 | 九江牧威利元科技中心（普通合伙） | A kind of antibacterial peptide and its application |
| CN109536507A (en) * | 2018-12-13 | 2019-03-29 | 海南大学 | The peptide and prokaryotic expression preparation method of Bu Shi silvery pomfret Scad antibacterial peptide gene and its coding |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483247A (en) * | 2015-12-29 | 2016-04-13 | 云南农业大学 | Method for judging disease resistance of Yunnan native chicken based on LEAP-2 (liver expressed antimicrobial peptide 2) gene |
| CN108314722A (en) * | 2018-04-27 | 2018-07-24 | 九江牧威利元科技中心（普通合伙） | A kind of antibacterial peptide and its application |
| CN109536507A (en) * | 2018-12-13 | 2019-03-29 | 海南大学 | The peptide and prokaryotic expression preparation method of Bu Shi silvery pomfret Scad antibacterial peptide gene and its coding |
Non-Patent Citations (2)
| Title |
|---|
| NP_001001606.1;无;《GenBank》;20180521;全文 * |
| 鸡内源性抗菌肽LEAP-2的生物信息学分析;杭柏林等;《江苏农业科学》;20181231;第46卷(第6期);46-50页 * |
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