CN111560059B - Antibacterial peptide-SYPU 2 and its mutant and its derivative and analogue - Google Patents
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Abstract
The invention relates to the technical field of biological medicines, and relates to antibacterial peptide-SYPU 2 and mutant ASK, GQ and RQ structures thereof, an obtaining method and application thereof, in particular to antibacterial peptide-SYPU 2 and structures of mutants, derivatives, analogs and active fragments thereof, a preparation method thereof, and application thereof as antibacterial drugs in the field of medicines. The antibacterial peptide-SYPU 2 has the sequence shown in SEQ ID No: 1, and the sequences of the mutant ASK, GQ and RQ are shown as SEQ ID No: 2-4. The antibacterial peptide-SYPU 2 and mutants ASK, GQ and RQ thereof, and derivatives, analogues and active fragments of the antibacterial peptide-SYPU 2 thereof have obvious antibacterial activity and can be used for preparing antibacterial drugs.
Description
The technical field is as follows:
the invention relates to the technical field of biological medicines, and relates to antibacterial peptide-SYPU 2 and mutant ASK, GQ and RQ structures thereof, an obtaining method and application thereof, in particular to antibacterial peptide-SYPU 2 and structures of mutants, derivatives, analogs and active fragments thereof, a preparation method thereof, and application thereof as antibacterial drugs in the field of medicines.
Background art:
the antibacterial peptide has the characteristics of small molecular weight, good water solubility, strong heat resistance, no immunogenicity, wide antibacterial spectrum, no drug resistance, unique action mechanism and the like, and is considered to be the first choice drug for replacing antibiotics. However, the extraction of the antibacterial peptide from natural resources has high cost, low yield, complicated process, and expensive chemical synthesis, and is difficult to apply and produce, so the production of the antibacterial peptide by using genetic engineering technology and protein extraction, separation and purification technology has important significance.
The scorpion, as a precious herb in traditional Chinese medicine, is also called whole worm and scorpion, and is listed in Kai Bao Ben Cao (materia Medica of materia Medica), listed in 40 volumes of the section of worms, and has more than 2000 years of medicinal history today, and has the efficacies of calming wind, relieving spasm, counteracting toxic pathogen, dissipating stagnation, dredging collaterals and relieving pain. The main active component of scorpion is toxin secreted by the tail gland, which contains a large amount of active components with pharmaceutical value, and is a natural active peptide library rich in various functional peptides.
At present, the number of components having antibacterial activity, such as scorpion analgesic and antibacterial active peptides, which are separated, purified and structurally analyzed from scorpions or scorpion venom is not large.
The invention content is as follows:
one of the objects of the present invention is to isolate and purify an antibacterial peptide-SYPU 2 having the structure of a polypeptide having an antibacterial activity from scorpion venom and to analyze the structure.
The second purpose of the invention is to realize the expression, separation and purification of the antibacterial peptide-SYPU 2 by using a genetic engineering technology. The method for obtaining the recombinant antibacterial peptide-SYPU 2 is simple and easy to implement.
The third purpose of the invention is the mutant of antibacterial peptide-SYPU 2, which is realized by respectively carrying out site-directed mutagenesis and in-vitro bioactivity experiment on antibacterial peptide-SYPU 2 molecules by utilizing the genetic engineering technology:
the antibacterial peptide-SYPU 2 has the sequence shown in SEQ ID No: 1, the amino acid sequence of which is:
VKDGYIADDRNCPYFCGRNAYCDGECKKNRAESGYCQWASKYGNACWCYKLPDDARIMKPGRCNGG。
on the basis of the antibacterial peptide-SYPU 2, the following mutants are obtained by further mutating different positions of the antibacterial peptide:
(1) the mutant ASK is obtained by deleting 37 th, 38 th, 39 th, 40 th and 41 th residues from the N-terminal of antibacterial peptide-SYPU 2, and the amino acid sequence of the mutant ASK is shown as SEQ ID No: 2 is shown in the specification;
(2) a mutant GQ with GT two amino acid residues inserted between 18 th to 19 th residues of antibacterial peptide-SYPU 2 and 5 th residues in 37 th to 41 th residues deleted, and the amino acid sequence of the mutant GQ is shown as SEQ ID No: 3 is shown in the specification;
(3) the amino acid sequence of the scorpion venom active peptide mutant RQ is shown in SEQ ID No.4, wherein RP amino acid residues are inserted between 18 th and 19 th residues of antibacterial peptide-SYPU 2, and 5 th residues in the 37 th to 41 th positions are deleted.
The expression, separation and purification of ASK, GQ and RQ of the antibacterial peptide-SYPU 2 mutant are realized by utilizing a genetic engineering technology. The recombinant antibacterial peptide-SYPU 2 mutant has simple and easy obtaining method.
The fourth purpose of the invention is to provide the application of the antibacterial peptide-SYPU 2 and the mutant, the derivative, the analogue and the active fragment thereof in preparing antibacterial drugs.
The antibacterial peptide-SYPU 2 and its mutant ASK, GQ or RQ of the invention form a pharmaceutical composition with one or more pharmaceutically acceptable carriers or excipients.
The present invention can prepare pharmaceutical compositions suitable for parenteral administration according to the basic principles and methods known in the pharmaceutical industry, and the pharmaceutical compositions of the present invention can be administered by various routes of administration, particularly, parenteral routes such as intravenous, intramuscular, intra-articular, intraperitoneal, intranasal, intradermal, subcutaneous, etc.
The antibacterial peptide-SYPU 2 and its mutant ASK, GQ and RQ can be obtained by the following method with reference to patent CN 201610210622:
(1) constructing an expression vector by using a gene recombination technology;
(2) performing expression in a host cell;
(3) separating and purifying the expression product from the expression engineering cell by affinity chromatography;
(4) cutting off the affinity purification tag in the expression product by using proteolytic enzyme;
(5) ultrafiltering to remove salt and concentrating to obtain recombinant antibacterial peptide-SYPU 2 or its mutant or its derivative or its analogue or its active fragment.
In particular, the amount of the solvent to be used,
the invention realizes the gene expression and acquisition of the antibacterial peptide-SYPU 2 and mutants, derivatives, analogues and active fragments thereof by the following technical scheme, which comprises the following steps:
the method comprises the steps of recombining genes encoding antibacterial peptide-SYPU 2 or mutants, derivatives, analogues and active fragments into an expression vector;
the recombinant expression vector is transformed or transfected into a host cell;
culturing the transformed host cell in the second step under a suitable induced expression condition;
the obtained expression product is harvested and purified.
The invention provides a method for separating and purifying the expression product of the antibacterial peptide-SYPU 2. The desired expression product can be isolated and purified from the lysate and culture medium of the engineered cells using methods such as metal ion chelating affinity chromatography, ultrafiltration, and the like. In the process of separating and purifying the expression product, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) or WESTERN blotting assay (WESTERN) can be used for detecting the existence of the expression product and the corresponding molecular size.
In order to obtain the high-purity recombinant antibacterial peptide-SYPU 2 or and mutant or derivative or analogue or active fragment thereof, the high-purity recombinant antibacterial peptide-SYPU 2 or mutant or derivative or analogue or active fragment thereof can be obtained by further purifying by cation exchange chromatography or anion exchange chromatography or hydrophobic chromatography or reverse chromatography, removing salt and hybrid protein by ultrafiltration and concentrating.
In addition, the invention also aims to provide application of the antibacterial peptide-SYPU 2 and mutants, derivatives, analogs and active fragments thereof in producing antibacterial drugs, and the antibacterial peptide-SYPU 2 and the mutants, derivatives, analogs and active fragments thereof or a pharmaceutical composition containing the antibacterial peptide-SYPU 2 and the mutants, derivatives, analogs and active fragments thereof can be used as a therapeutic agent for treating various infection-related diseases of human bodies. The therapeutically effective dose of the pharmaceutical composition of the present invention will generally be determined by the clinician on an individualized basis, depending on the nature, severity and susceptibility to the disease, and the route of administration, among other factors.
Description of the drawings:
FIG. 1 shows the primary structure of antimicrobial peptide-SYPU 2;
FIG. 2 shows the bacteriostatic action of the antibacterial peptide-SYPU 2 and its mutant on Pseudomonas aeruginosa;
FIG. 3 shows the bacteriostatic action of the antibacterial peptide-SYPU 2 and its mutant on Escherichia coli;
FIG. 4 shows the bacteriostatic action of the antibacterial peptide-SYPU 2 and its mutant on Staphylococcus aureus.
The specific implementation mode is as follows:
the following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the scope of the claims appended hereto in any way.
Example 1:
antibacterial peptide-SYPU 2
The method comprises the steps of taking scorpions or scorpion venom as a raw material, separating and purifying each component by using a conventional column chromatography technology, determining which component has antibacterial activity by combining with a common antibacterial activity experimental result, further separating and purifying the active component by using column chromatography, separating and purifying the active component by using column chromatography for multiple times and screening the antibacterial activity of the component to obtain the antibacterial peptide from the scorpion venom, and analyzing the active peptide-antibacterial peptide-SYPU 2 by using a conventional method, wherein the structure of the active peptide-antibacterial peptide-SYPU 2 is shown as SEQ ID No. 1.
Example 2:
obtaining of recombinant antibacterial peptide-SYPU 2
1. Construction of antibacterial peptide-SYPU 2 gene expression vector
This example illustrates the construction strategy and basic methodology for the expression of the gene for the antimicrobial peptide-SYPU 2 of the present invention and its mutants, derivatives, analogs, active fragments.
Designing corresponding oligonucleotide primers F1 and R1 according to the N-terminal amino acid sequence and the C-terminal amino acid sequence of the antibacterial peptide-SYPU 2, and adding the sequences of the hydrolysis sites of restriction endonucleases Nco I and BamH I to the 5' ends of the two oligonucleotide primers; PCR amplification is carried out by taking scorpion cDNA as a template, products are detected by agarose gel electrophoresis, and gel recovery of nucleic acid fragments is carried out; after double digestion by restriction endonucleases Nco I and BamH I, the plasmid subjected to double digestion is recombined and connected under the action of T4 DNA ligase, escherichia coli competent cells DH 5 alpha are thermally transformed, positive transformants are obtained by colony PCR and restriction endonuclease digestion verification screening, and then the positive transformants are submitted to a biotechnology service company for DNA sequence determination. The antibacterial peptide-SYPU 2 gene is obtained by the genetic engineering method.
2. Obtaining of recombinant antibacterial peptide-SYPU 2
The antibacterial peptide-SYPU 2 gene is recombined into expression plasmid by using gene engineering technology, and the plasmid is extracted for sequencing.
(1) Expression of antibacterial peptide-SYPU 2
The positive recombinant plasmid was transformed into E.coli BL21 (. lamda.DE 3) by heat, and then a single colony was picked from the LB solid plate and inoculated into 3ml of LB (containing antibiotic, 50. mu.g/ml) and cultured overnight at 37 ℃ under shaking at 200 r/min. Inoculating the overnight culture into 400ml of a triangular flask containing a fresh LB culture medium containing the corresponding antibiotic according to the inoculation amount of 1%, carrying out shaking culture at 37 ℃ and 200r/min until the OD600 is 0.6-0.8, adding an inducer isopropyl beta-D-thiogalactoside (IPTG) or an expression vector expression inducer with the final concentration of 0.166mmol/L or an expression vector expression condition to realize expression, and culturing for 4h to induce the expression of the target gene.
(2) Sample pretreatment
After the fermentation is finished, 3500g and 4 ℃ are centrifuged for 20min, and the thalli are collected. Resuspending the cells in 40ml lysis buffer (0.1M PBS,0.5M NaCl, pH8.0), sonicating, centrifuging at 12,000g at 4 ℃ for 20min after sonication to obtain a supernatant, repeating the disruption of the obtained pellet once according to the above steps, and combining the supernatants.
(3) Metal ion chelating affinity chromatography separation of recombinant antibacterial peptide-SYPU 2
Directly loading to a metal ion chelating chromatographic column pre-equilibrated with 0.1M PBS (pH 8.0), washing with four stages of pH buffers for 5 bed volumes, eluting with 0.1M PBS (containing 1M NaCl, pH 3.0), and collecting the eluate. The purity of the product was verified by 12.5% SDS-PAGE. The high-efficiency expression of the recombinant protein is realized by the column chromatography chromatogram and the SDS-PAGE atlas, so that the electrophoretic purity is achieved.
(4) Ultrafiltration treatment of recombinant antibacterial peptide-SYPU 2
The purpose of the ultrafiltration treatment was to remove salts or hetero-proteins from the elution solution or to replace the buffer, and in addition, to concentrate the recombinant antimicrobial peptide-SYPU 2 solution. And selecting an ultrafiltration membrane capable of intercepting the recombinant antibacterial peptide-SYPU 2 for ultrafiltration treatment, collecting an ultrafiltration concentrated solution (the recombinant antibacterial peptide-SYPU 2 is intercepted), and permeating salt and buffer solution buffer pairs, and the foreign proteins and water which can permeate the ultrafiltration membrane, so as to realize the purposes of removing salt and foreign proteins or replacing buffer solution or concentrating.
Characterization of the ultrafiltration treatment: 1. the ultrafiltration membrane for retaining the recombinant antibacterial peptide-SYPU 2 is selected from ultrafiltration membrane with molecular weight of 1kDa or 2kDa or 3kDa or 5kDa, preferably ultrafiltration membrane with molecular weight of 3 kDa; 2. the temperature of ultrafiltration treatment is selected to be 4 ℃, and the condition does not damage the physicochemical property of the ultrafiltration membrane and does not influence the activity of the recombinant antibacterial peptide-SYPU 2.
(5) Preparation of high-purity recombinant antibacterial peptide-SYPU 2
And (3) purifying the recombinant antibacterial peptide-SYPU 2 purified in the step (4) by using cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography or reverse chromatography, removing salts and foreign proteins by ultrafiltration, and concentrating to obtain the high-purity recombinant antibacterial peptide-SYPU 2.
Similarly, according to the conventional technology of genetic engineering, the antibacterial peptide-SYPU 2 recombinant expression plasmid is transfected into a yeast expression host cell, an engineering cell for expressing a target gene is screened, and the expression of the target gene is realized according to a normal technical system. Purifying by cation exchange chromatography or anion exchange chromatography or hydrophobic chromatography or reverse chromatography, ultrafiltering to remove salt and foreign protein, and concentrating to obtain high purity recombinant antibacterial peptide-SYPU 2.
Example 3:
obtaining of recombinant antibacterial peptide-SYPU 2 mutant
This example shows the strategy and basic procedure for obtaining recombinant antibacterial peptide-SYPU 2 mutant, similar to the strategy and basic procedure of example 2.
This example illustrates the strategy and basic methodology for constructing recombinant expression plasmids for expressing the ASK mutant of the present invention, the antimicrobial peptide-SYPU 2.
Corresponding oligonucleotide primers are designed according to the amino acid sequence of the antibacterial peptide-SYPU 2, and the aim of deleting codons of 5 amino acid residues in the 37 th to 41 th positions in the antibacterial peptide-SYPU 2 molecule is fulfilled by means of PCR and gene recombination technology. The site-directed mutagenesis of the ASK gene of the antibacterial peptide-SYPU 2 mutant was performed in the same manner as in example 2 to construct expression plasmids, induce expression, purify expression products, and the like.
The antibacterial peptide-SYPU 2 mutant ASK (deletion of 37 th to 41 th residues) has a structure shown in SEQ ID No.2 and is characterized in that:
VKDGYIADDRNCPYFCGRNAYCDGECKKNRAESGYCYGNACWCYKLPDDARIMKPGRCNGG。
the antibacterial peptide-SYPU 2 mutant GQ (GT is inserted between 18-19 residues, 5 amino acid residues are deleted from 37 th-41 th positions) is obtained in the same way, the structure of the antibacterial peptide is shown in SEQ ID No.3, and the antibacterial peptide-SYPU 2 mutant GQ is characterized in that:
VKDGYIADDRNCPYFCGRGTNAYCDGECKKNRAESGYCYGNACWCYKLPDDARIMKPGRCNGG。
the antibacterial peptide-SYPU 2 mutant RQ (RP is inserted between 18-19 residues, and 5 amino acid residues are deleted from 37 th-41 th positions) is obtained by the same principle, and the structure of the mutant RQ is shown as SEQ ID No.4 and is characterized in that:
VKDGYIADDRNCPYFCGRRPNAYCDGECKKNRAESGYCYGNACWCYKLPDDARIMKPGRCNGG。
example 4:
obtaining of recombinant antibacterial peptide-SYPU 2 derivative, analogue and active fragment
The N-terminal of antibacterial peptide-SYPU 2 or antibacterial peptide-SYPU 2 mutant is added with
MGSSHHHHHHSSGLVPRGSHMG or MGSSHHHHHHSSGLVPRGSGGGG, and is recombinant antibacterial peptide-SYPU 2 derivative, analog or active fragment.
This example shows the strategy and basic method for obtaining derivatives, analogs and active fragments of antibacterial peptide-SYPU 2, similar to those of example 2 and example 3.
Example 5:
antibacterial effect of antibacterial peptide-SYPU 2 and its mutant, derivative, analogue and active fragment
1. Enzyme-linked turbidimetry
(1) Activation of the Strain
Three standard strains, namely staphylococcus aureus (G + bacteria), pseudomonas aeruginosa (G-bacteria) and escherichia coli (G-bacteria) are inoculated into 3ml of LB culture medium according to the proportion of 1:100 and cultured for 12h at 37 ℃. The bacterial liquid is further inoculated into 4ml of LB culture medium and cultured for 12h at 37 ℃.
(2) Preparation of the bacterial suspension
Enzyme-labeled measurement of blank plate OD595The value is determined by adding 200. mu.l of 0.5 McLeod turbidimetric solution to the ELISA plate595And measuring the value, and taking an average value for ten times.
And secondly, subpackaging the activated bacteria liquid into 2 Ep tubes, and centrifuging to remove the supernatant. One tube was added with 2ml of sterile physiological saline and mixed well.
Thirdly, 200 mul of physiological saline resuspension bacterial liquid is added into the ELISA plate to determine OD595The value is obtained. Diluting the bacterial liquid to 0.5 McLeod turbidity solution OD595The same value, the bacterial concentration is about 1X 108cfu/ml, the bacterial solution was diluted 100 times to a bacterial concentration of about 1X 106cfu/ml was used as the concentration of bacteria used in the experiment and the dilution factor at this time was recorded.
And (iv) diluting the bacterial suspension into bacterial suspension with the same times by using LB culture medium according to the dilution times of the physiological saline.
(3) Culture of bacterial liquid
Physiological saline is used as a negative control, 0.5 mu g/ml kanamycin is used as a positive control, and the recombinant active peptide is used as a sample to be detected (the final concentration is 0.3 mg/ml). Adding 100 μ l physiological saline and 100 μ l LB culture medium into negative control, adding 100 μ l kanamycin and 100 μ l bacterial suspension into positive control, adding 100 μ l bacterial suspension and 100 μ l protein sample into test group, culturing at 37 deg.C for 6 hr, and determining OD595Values, three parallel runs were performed for each group.
2. Antibacterial peptide-SYPU 2 and its mutant bacteriostatic activity
In the embodiment, the biological activity, namely the bacteriostatic activity, of the antibacterial peptide-SYPU 2 and the mutant thereof is determined by an enzyme-labeled turbidimetry method.
As can be seen from the results in FIG. 2, the antibacterial peptide-SYPU 2 and its mutant ASK and mutant RQ have bacteriostatic action on Pseudomonas aeruginosa.
As can be seen from the results in FIG. 3, the ASK mutant and the GQ mutant of the antibacterial peptide-SYPU 2 have bacteriostatic action on Escherichia coli.
As shown in the result of FIG. 4, the antimicrobial peptide-SYPU 2 mutant ASK and the mutant GQ have bacteriostatic action on staphylococcus aureus.
In the figure denotes p < 0.05; denotes p < 0.01; p < 0.0005; denotes p < 0.0001.
3. Antibacterial peptide-SYPU 2 derivative, analogue and active fragment with antibacterial activity
In this example, the biological activity, i.e., the bacteriostatic activity, of the derivative, the analogue and the active fragment of the recombinant antibacterial peptide-SYPU 2 obtained in example 4 was examined by an enzyme-labeled turbidimetric method.
The test result shows that:
in the pseudomonas aeruginosa test group: the derivative, the analogue and the active fragment of the recombinant antibacterial peptide-SYPU 2 obtained in the embodiment 4 have bacteriostatic action; there was no significant difference between the antibacterial peptide-SYPU 2 and its mutant ASK, mutant RQ, respectively, at the same concentration.
In the staphylococcus aureus test group: the derivative, the analogue and the active fragment of the recombinant antibacterial peptide-SYPU 2 obtained in the embodiment 4 have bacteriostatic action; compared with the antibacterial peptide-SYPU 2 mutant ASK and the mutant GQ which correspond to the same concentration, the two have no significant difference.
In the E.coli test group: the derivative, the analogue and the active fragment of the recombinant antibacterial peptide-SYPU 2 obtained in the embodiment 4 have bacteriostatic action; compared with the antibacterial peptide-SYPU 2 mutant ASK and the mutant GQ which correspond to the same concentration, the two have no significant difference.
Sequence listing
<110> Shenyang university of pharmacy
<120> antibacterial peptide-SYPU 2 and its mutant and its derivative and analogue
<130> antibacterial peptide-SYPU 2 and its mutant and its derivative and analogue
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Claims (9)
1. The antibacterial peptide-SYPU 2 mutant is characterized in that the amino acid sequence is shown as SEQ ID number 2.
2. An analog of the antibacterial peptide-SYPU 2 mutant, wherein MGSSHHHHHHSSGLVPRGSHMG or MGSSHHHHHHSSGLVPRGSGGGG is added before the N-terminal of the antibacterial peptide-SYPU 2 mutant.
3. A method for producing the antibacterial peptide-SYPU 2 mutant according to claim 1 or the analogue of antibacterial peptide-SYPU 2 mutant according to claim 2, comprising:
(1) constructing an expression vector by using a gene recombination technology;
(2) performing expression in a host cell;
(3) separating and purifying the expression product from the expression engineering cell by affinity chromatography;
(4) cutting off the affinity purification tag in the expression product by using proteolytic enzyme;
(5) ultrafiltering to remove salt and concentrating to obtain recombinant antibacterial peptide-SYPU 2 mutant or antibacterial peptide-SYPU 2 mutant analogue.
4. The process according to claim 3, wherein the purification comprises (1) purification by cation exchange chromatography, (2) purification by anion exchange chromatography and purification by cation exchange chromatography, (3) purification by hydrophobic chromatography, (4) purification by reverse phase chromatography, (5) removal of salts and hetero-proteins by ultrafiltration and concentration to obtain a high-purity recombinant antibacterial peptide-SYPU 2 mutant or an analog of antibacterial peptide-SYPU 2 mutant.
5. The antibacterial peptide-SYPU 2 mutant according to claim 1 or the analog of antibacterial peptide-SYPU 2 mutant according to claim 2, which is obtained by chemical synthesis, wherein the chemical synthesis is artificial synthesis or synthesizer synthesis.
6. A pharmaceutical composition comprising the antibacterial peptide-SYPU 2 mutant of claim 1 or an analog of the antibacterial peptide-SYPU 2 mutant of claim 2.
7. Use of the antibacterial peptide-SYPU 2 mutant of claim 1 or an analogue of the antibacterial peptide-SYPU 2 mutant of claim 2 or the composition of claim 6 in the preparation of a bacteriostatic medicament.
8. The use of claim 7, wherein the bacteria are Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus.
9. Use according to claim 7 or 8, characterized in that: the antibacterial peptide-SYPU 2 mutant or analogue or the composition thereof is mixed with a pharmaceutically acceptable carrier to prepare clinically acceptable injections, oral preparations, transdermal absorption preparations and mucosal absorption preparations.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985467A (en) * | 2010-09-26 | 2011-03-16 | 沈阳药科大学 | Analgesic active peptide VGG and preparation method and applications thereof |
| CN102690342A (en) * | 2011-03-22 | 2012-09-26 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
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| CN101985467A (en) * | 2010-09-26 | 2011-03-16 | 沈阳药科大学 | Analgesic active peptide VGG and preparation method and applications thereof |
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