CN109266655B - Antibacterial peptide and prokaryotic expression method and application thereof - Google Patents
Antibacterial peptide and prokaryotic expression method and application thereof Download PDFInfo
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- CN109266655B CN109266655B CN201811038571.9A CN201811038571A CN109266655B CN 109266655 B CN109266655 B CN 109266655B CN 201811038571 A CN201811038571 A CN 201811038571A CN 109266655 B CN109266655 B CN 109266655B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses an antibacterial peptide and a prokaryotic expression method and application thereof. The invention adopts a prokaryotic expression vector to construct a recombinant vector containing the coding gene of the antibacterial peptide cecropin-BM, realizes the prokaryotic expression of the antibacterial peptide cecropin-BM, has simple and easy operation, and has obvious in-vitro antibacterial effect without any treatment after the purification of the obtained recombinant antibacterial peptide.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an antibacterial peptide, and a prokaryotic expression method and application thereof.
Background
The antibacterial peptide (antimicrobial peptide) is a small molecular polypeptide naturally existing in almost all organisms, is an important component of natural immunity of the organisms, not only has broad-spectrum antibacterial effect, but also has killing effect on some fungi, parasites and viruses with partial envelopes. Different from the conventional antibiotics, the antibacterial peptide is not easy to cause the generation of drug resistance of bacteria, has a killing effect on antibiotic resistant strains, and shows great potential in the field of development of novel antibacterial drugs. However, natural antibiotics also have the defects of limited sources, low titer, high artificial synthesis cost caused by long polypeptide sequences and the like. Therefore, it is of great importance to develop and prepare a new generation of antibacterial peptides capable of overcoming the above-mentioned disadvantages.
The natural antibacterial peptide is mostly cationic antibacterial peptide (the charge number is between +2 and + 9), and can form a stable alpha-helical structure. The alpha-helix can be positioned at the N-end or the C-end of the peptide chain, has water-lipid amphipathy, and is the basis of conformation of the antibacterial peptide for exerting antibacterial activity. It is believed that the antibacterial peptide with positive charge is adsorbed to the surface of the bacterial membrane with negative charge through the action of electrostatic attraction, and then forms an amphipathic helical conformation, and the amphipathic helical conformation is helpful for the hydrophobic surface of the antibacterial peptide to be inserted into a cell membrane phospholipid bilayer, so that the bacterial membrane is damaged, and the bactericidal effect is achieved. It has been shown that increasing the hydrophobicity and net positive charge of an antimicrobial peptide in a certain range enhances its bactericidal activity, because increasing the hydrophobicity enhances the ability of the hydrophobic group of the antimicrobial peptide to insert into the bacterial plasma membrane, and increasing the net charge enhances the ability of the antimicrobial peptide to bind to the bacterial plasma membrane. In addition, increasing the hydrophobicity and net charge number is also more beneficial to increase the stability of the alpha-helix.
Cecropin A is the first cationic antimicrobial peptide found in insects, and the precursor peptide is 61 amino acids. Cathelicidins antibacterial peptides are the predominant cationic antibacterial peptides in mammalian endogenous antibacterial peptides, BMAP-27 is representative of the Cathelicidins family, and the mature peptide consists of 129 amino acid residues. In order to obtain the novel antibacterial peptide with strong antibacterial activity and shorter peptide chain, the invention takes natural cecropin A and BMAP-27 as blueprints, designs a cationic antibacterial peptide consisting of 32 amino acid residues on the basis of analyzing physicochemical properties of alpha-helix, net positive charge number, hydrophobicity, amphipathy and the like of the two peptides, and establishes a prokaryotic expression method of the novel antibacterial peptide. The result of bacteriostatic test shows that the antibacterial peptide has good antibacterial activity.
Disclosure of Invention
The invention aims to provide an antibacterial peptide, a prokaryotic expression method and application thereof. The antibacterial peptide is a novel cationic antibacterial peptide and is composed of 32 amino acid residues, and the recombinant antibacterial peptide obtained by the prokaryotic expression method has an obvious in-vitro antibacterial effect without any treatment after purification.
In order to achieve the purpose, the invention adopts the following technical scheme:
the nucleotide sequence of the coding gene of the antibacterial peptide cecropin-BM is shown as SEQ ID No. 1.
Preferably, the primer sequences for PCR amplification of the expression vector are 8:
P1：CGCGGATCCATGAGCTATGGCCAGGGCCAGTTTTTTCGTGAAATTGAAAATCTGAAAGAATATTTCA；
P2：CCGCCTTTCGCCACATCCGGGCTGCTCGCGTTGAAATATTCTTTCAGATTTTCAATTTCA；
P3：GATGTGGCGAAAGGCGGTCCTCTGTTTAGCGAAATTCTGAAAAATTGGAAAGATGAAAGC；
P4：AAGCTCACAACTGGCTCTGAATAATCTTTTTATCGCTTTCATCTTTCCAATTTTTCAGA；
P5：CAGAGCCAGATTGTGAGCTTTTACTTCAAACTGTTTGAGAACTTGAAAGATGAGGAGTAA；
P6：CGCAAACACAAAGCTCAGAATACGCACAAATGTCATTACTCCTCATCTTTCAAGTTCTCA；
P7：ATTCTGAGCTTTGTGTTTGGCTGGTGCTGGCGCTGCGTTTTAAGCGTTTTCGTAAAAAG；
P8：CGGAATTCGCTCAGCTTCTTAAACAGTTTTTTGAACTTTTTACGAAAACGCTTAAAACG。
the invention also provides a recombinant vector of the antibacterial peptide cecropin-BM, which comprises a coding gene of the cecropin-BM shown as SEQ ID No. 1.
The invention also provides an antibacterial peptide cecropin-BM which is coded by the nucleotide sequence shown in SEQ ID No. 10.
The invention also provides a method for expressing the cecropin-BM pronucleus of the antibacterial peptide, which comprises the following steps:
obtaining a coding gene of an antibacterial peptide cecropin-BM;
constructing a recombinant vector: connecting a PCR amplification product of a coding gene of the antibacterial peptide cecropin-BM to a pMD19-T cloning vector to construct a recombinant plasmid pMD 19-IFN-cecropin-BM; respectively carrying out enzyme digestion on the recombinant plasmid pMD19-IFN-cecropin-BM and a prokaryotic expression vector pET-28a (+), and constructing a recombinant vector pET28-IFN-cecropin-BM containing a coding gene of the cecropin-BM;
expression of the antimicrobial peptide cecropin-BM: the recombinant vector pET28-IFN-cecropin-BM is transformed into BL21 strain, and bacteria containing the antibacterial peptide cecropin-BM are collected after IPTG induction.
Preferably, the method further comprises: treating the centrifugally crushed cell bacterium liquid by using PBS (phosphate buffer solution) heavy suspension containing urea and imidazole, centrifuging and collecting precipitates to obtain supernatant;
and purifying the supernatant by adopting a Ni column affinity chromatography to obtain the purified antibacterial peptide cecropin-BM.
The invention also provides application of the antibacterial peptide cecropin-BM in inhibiting the growth of escherichia coli and staphylococcus aureus.
The invention has the following technical characteristics:
the invention adopts a prokaryotic expression vector to construct a recombinant vector containing the coding gene of the antibacterial peptide cecropin-BM, realizes the prokaryotic expression of the antibacterial peptide cecropin-BM, has simple and easy operation, and has obvious in-vitro antibacterial effect without any treatment after the purification of the obtained recombinant antibacterial peptide.
Drawings
FIG. 1 prediction of Cerropin-BM amphiphilicity.
FIG. 2 SOE-PCR gel electrophoresis results.
FIG. 3 shows the results of Tricine-SDS-PAGE gel electrophoresis (1: expression of whole protein from pET-Cecropin-BM plasmid, 2: expression of whole protein from pET-28a (+) plasmid, 3: expression of purified pET-Cecropin-BM plasmid, and 4: expression of purified pET-28a (+) empty plasmid).
FIG. 4 shows the recombinant protein inhibition test (A: E. coli, B: Staphylococcus aureus; 1: kanamycin (100. mu.g); 2: Cecropin-BM (100. mu.g); 3: Cecropin-BM (50. mu.g); 4: Cecropin-BM (25. mu.g); 5: pET-28a (+) empty vector plasmid control)
Detailed Description
The following specific examples are further illustrative of the methods and techniques provided by the present invention and should not be construed as limiting the invention thereto.
First, the materials and sources used in the embodiments of the present invention are as follows:
1 bacterial species and plasmids
DH5 alpha chemical component Cell (New Biotechnology Co., Ltd., Beijing Optimus department); BL21(DE3) chemical company Cell (Shanghai Weidi Biotechnology Co., Ltd.); pMD19-T Vector (TaKaRa Co.); pET-28a (+) vector, E.coli, Staphylococcus aureus were all from this laboratory storage.
2 reagent
High fidelity enzyme PrimeSTAR HS DNA Polymerase, Taq enzyme, agarose gel electrophoresis 10 XLoading Buffer, DL1000 DNA Marker, restriction endonuclease EcoR I and BamH I, plasmid connection DNA Ligation Kit, and nickel column His60 Ni quality Columns used for purifying protein are all products of TaKaRa company; the AxyPrep plasmid DNA small-scale kit is a product of Axygen company; the PCR product purification kit and the BCA protein quantitative kit are purchased from Melam biology company;
protein Marker used by Tricine-SDS-PAGE is a Fred biological product; Tricine-SDS-PAGE protein loading buffer (5X) was the product of Beyotime.
Secondly, the prokaryotic expression method of the antibacterial peptide cecropin-BM in the specific embodiment of the invention is as follows:
1 design and physicochemical property prediction of novel antibacterial peptide cecropin-BM
The secondary structures of cecropin A (accession number: NP-001037462) and BMAP-27 (accession number: NP-001037462) were analyzed using the online tool SOPMA (https:// npsa-prabi. ibcp. fr /), to obtain the alpha-helical amino acid sequences in both peptide chains. The alpha-helical fragments from the two peptides were selected for combination, the charge number and hydrophobicity of the combined peptide fragments were analyzed using the on-line tool ExPASY (https:// web. ExPASy. org/protparam /), and the amphiphilicity prediction was performed using the Antiprot 6.9.3 software. The amino acid sequence of the new antibacterial peptide is determined by integrating the parameters of alpha-helix, charge, hydrophobicity, amphipathy and the like, and the new antibacterial peptide is named cecropin-BM.
Prokaryotic expression of 2 Cecropin-BM
2.1 nucleotide sequence design
Expressing cecropin-BM by using bicistronic expression vector, using 21-85 amino acid sequence of IFN-gamma (NP-776511) as first cistron, and using cecropin-BM as second cistronThe amino acid sequence serves as the second cistron. After gene modification according to the codon preference of escherichia coli, the two gene sequences are connected by 5'-GAGGAGTAATGACA-3', and the obtained IFN-cecropin-BM gene sequences are combined as follows:
(Single underlined is the first cistron, bold letters are the joining sequence, double underlined is the second cistron sequence).
2.2 PCR amplification
8 primers are designed, BamH I restriction enzyme sites and EcoRI restriction enzyme sites are respectively introduced into the 5' -ends of the first primer and the last primer, and IFN-cecropin-BM gene is synthesized by an overlap extension PCR method. The primers were synthesized by Hangzhou Optingke Biotechnology Limited (Table 1).
PCR amplification is carried out in 2 steps: firstly, adding primers P1-P4 and P5-P8 into a 20 mu L PCR system respectively, and amplifying to obtain two PCR products which are named as P14 and P58 respectively; and secondly, adding PCR products P14 and P58 into a 20 mu L PCR system, and amplifying the full-length gene of IFN-Cecropin-BM by taking P1 and P8 as upstream and downstream primers. The reaction system is shown in Table 2. The PCR reaction conditions are as follows: 5min at 94 ℃ (30 s at 98 ℃, 5s at 57 ℃ and 15 s-1 min at 72 ℃) multiplied by 25 cycles, and 10min at 72 ℃ extension. The PCR product was detected by electrophoresis on a 1.5% agarose gel.
TABLE 1 PCR primers
Note: underlined is the restriction enzyme site
TABLE 2PCR reaction System
2.3 construction of pMD19-IFN-cecropin-BM plasmid
The PCR product purification kit is adopted to purify the cecropin-BM product, Taq enzyme is utilized to carry out A-tail addition reaction under the reaction conditions of 72 ℃ and 30min, and the reaction system is shown in Table 3. Subcloning cecropin-BM with A tail to pMD19-T vector, transforming DH5 alpha competent cell, selecting positive colony for amplification culture, and sending the bacterial liquid to Hippocastine Biotechnology Limited in Hangzhou department for sequencing.
TABLE 3 reaction system of cecropin-BM product with A tail
DH5 alpha with correct sequencing was expanded in LB liquid medium and plasmid DNA was extracted using AxyPrep plasmid DNA minikit. The extraction method is carried out according to the instruction.
2.4 construction of expression vector pET28-IFN-cecropin-BM
2.4.1 plasmid and vector double digestion
The pMD19-IFN-cecropin-BM plasmid and the pET-28a (+) vector were double digested with restriction enzymes BamH I and EcoRI, respectively, in 10 XK buffer, and digested at 37 ℃ overnight. Mixing the double digestion products of pMD19-IFN-cecropin-BM plasmid and pET-28a (+) vector according to the molar ratio of 10:1 (the total volume is 5 mu L), placing the mixture in a water bath at 65 ℃ for reaction for 2min, taking out the mixture, immediately carrying out ice bath for 2min, adding Solution I5 mu L, and connecting at 16 ℃ overnight.
2.4.2 plasmid transformation
pET28-IFN-cecropin-BM plasmid is added into BL21 competence, and after being set aside in ice bath for 25min, the mixture is set aside in water bath at 42 deg.c for 45s and inserted into ice bath immediately for 2 min. Add 700. mu.L of antibiotic-free sterile LB medium and shake-culture at 37 ℃ and 200rpm for 60 min. And sucking 100 mu L of bacterial liquid, uniformly coating the bacterial liquid on the surface of a solid LB culture medium containing kanamycin, and culturing the solid LB culture medium in an incubator at 37 ℃ for 10-14 h. A single colony is picked up and inoculated in 3mL liquid LB culture medium containing kanamycin, and is cultured by shaking at 37 ℃ and 200rpm until the bacterial liquid is turbid, and then the bacterial liquid is sent to the department of engine for sequencing.
2.4.3 inducible expression of the protein of interest
Selecting positive transformant, inoculating to LB culture medium, shaking at 37 deg.C and 200rpm for overnight culture, inoculating 50 μ L bacterial liquid to 500mL liquid LB culture medium, shaking at 37 deg.C and 230rpm for culture to OD570Reaching 0.6, IPTG was added to a final concentration of 0.75mM, and expression was induced at 37 ℃ for 4 h. BL21(DE3) transformed with the empty pET28a (+) vector plasmid was used as a control.
After induction expression is finished, the thalli is collected by centrifugation (4 ℃, 10000g and 30min of centrifugation), the thalli is resuspended by PBS liquid containing 8M urea and 20mM imidazole, the thalli is broken by ultrasound (4 ℃, the power is 40%, the ultrasound is turned on for 2s and turned off for 4s, and 30min is totally turned off), 10000g and the supernatant is collected by centrifugation for 30 min.
2.4.4 Ni column affinity chromatography
Taking the supernatant, and purifying the target protein by adopting a Ni column affinity chromatography, wherein the method comprises the following specific steps: (1) the Ni column was rinsed with 5mL of binding buffer (containing 8M urea, 20mM imidazole); (2) adding 5mL of recombinant protein supernatant, and reversing and uniformly mixing to ensure that the protein is fully combined with the Ni column; (3) vertically placing the Ni column, completely flowing liquid in the column after the filler is settled at the bottom, and collecting the liquid after the column passes through; (4) repeating steps 2 and 3 until all supernatant is filtered; (5) adding 5mL of rinsing liquid (containing 8M urea and 40mM imidazole) into the column, reversing, uniformly mixing, standing, and after the filler is settled, completely draining the liquid in the column to ensure that the foreign protein is sufficiently eluted; (6) adding 1mL of eluent (containing 8M urea and 300mM imidazole) into the column, reversing the mixture from top to bottom, mixing the mixture evenly, standing the mixture, and fully collecting liquid in the column after filler is settled to obtain the target protein. Repeating the steps 1-6 to fully collect the target protein.
2.4.5 Tricine-SDS-PAGE gel electrophoresis
After the purified target protein is subjected to protein quantification by a BCA kit, electrophoresis is performed on Tricine-SDS-PAGE gel (4% concentrated gel, 10% spacer gel and 20% separation gel), Coomassie bright staining is performed, and the result is recorded by photographing.
3 recombinant protein antibacterial activity identification
The antibacterial activity identification is carried out by using an agarose plate diffusion method, and the used E.coli and staphylococcus aureus are the experimentsClinical isolates stored in the house. The method comprises the following specific steps: (1) respectively culturing E.coli and Staphylococcus aureus in liquid LB culture medium to logarithmic growth phase (OD)5700.4-0.6); (2) diluting the bacterial liquid by 10 times, respectively coating 50 mu L of bacterial liquid on a solid LB flat plate, culturing at 37 ℃ for 16h, and counting colonies; (3) bacterial liquid of E.coli and Staphylococcus aureus was diluted to 0.5 M.standard unit (1.5X 10)8CFU/mL), respectively and uniformly coating 50 mu L of bacterial liquid on an LB flat plate; (4) the above dishes were punched using a sterile punch and 25. mu.g, 50. mu.g and 100. mu.g of Ni column purified Cecropin-BM was added to each well. Kanamycin positive control wells (100. mu.g/well) and negative control wells (100. mu.g/well of pET-28a (+) empty plasmid expression product were added to each well), and the bacteriostatic effect was observed after culturing at 37 ℃ for 14 hours.
Third, physicochemical properties and antibacterial activity of antibacterial peptide cecropin-BM
1 prediction of physicochemical properties of recombinant protein Cecropin-BM
The bioinformatics analysis result shows that the cecropin-BM designed by the invention has higher alpha-helicity, the net charge number is +11, the protein is hydrophobic protein, and the property is stable (Table 4). The results of the amphipathy analysis showed that the cecropin-BM peptide chain had good amphiphilicity (FIG. 1).
TABLE 4 Cecropin-BM second level organization and prediction of physicochemical Properties
2PCR product gel electrophoresis results
As shown in FIG. 2, the size of the cecropin-BM gene fragment synthesized by overlap extension PCR was 320bp, which is consistent with the expected size. The sequencing result shows that the sequence is completely correct.
3 Tricine-SDS-PAGE electrophoresis result
The purified target protein formed 2 bands on Tricine-SDS-PAGE gel, the molecular weight size was 8.23kDa and 4.8kDa, respectively, and the sizes coincided with the expected molecular weights of IFN-His and cecropin-BM-His (FIG. 3), indicating that cecropin-BM was successfully expressed.
4 recombinant protein Cecropin-BM antibacterial activity test result
As shown in figure 4, after 25-100 μ g of recombinant cecropin-BM is added to the sample adding holes of E.coli and staphylococcus aureus culture plates, an obvious inhibition zone can be formed, the higher the addition amount is, the larger the inhibition zone is, and the pET-28a (+) empty plasmid expression product has no inhibition effect. The zone diameters formed by 100. mu.g of cecropin-BM on E.coli and Staphylococcus aureus agarose plates were 2.9cm and 2.8cm, respectively, while the zone diameters formed by 100. mu.g on E.coli and Staphylococcus aureus agarose plates were 3.2cm and 3.1cm, respectively (Table 5). The results show that the cecropin-BM of the invention has better in vitro bacteriostasis.
TABLE 5 results of Cecropin-BM bacteria inhibition test
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
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Claims (6)
1. A coding gene of antibacterial peptide cecropin-BM is characterized in that the nucleotide sequence of the coding gene is shown in SEQ ID No. 1.
2. A recombinant vector of an antibacterial peptide cecropin-BM, which is characterized by comprising a coding gene of the cecropin-BM shown as SEQ ID No. 1.
3. An antibacterial peptide cecropin-BM, which is encoded by a nucleotide sequence shown as SEQ ID No. 10.
4. A method for prokaryotic expression of an antibacterial peptide cecropin-BM, which is characterized by comprising the following steps:
obtaining a coding gene of an antibacterial peptide cecropin-BM; the nucleotide sequence of the coding gene is shown as SEQ ID No. 1;
constructing a recombinant vector: connecting a PCR amplification product of a coding gene of the antibacterial peptide cecropin-BM to a pMD19-T cloning vector to construct a recombinant plasmid pMD 19-IFN-cecropin-BM; respectively carrying out enzyme digestion on the recombinant plasmid pMD19-IFN-cecropin-BM and a prokaryotic expression vector pET-28a (+), and constructing a recombinant vector pET28-IFN-cecropin-BM containing a coding gene of the cecropin-BM;
expression of the antimicrobial peptide cecropin-BM: the recombinant vector pET28-IFN-cecropin-BM is transformed into BL21 strain, and bacteria containing the antibacterial peptide cecropin-BM are collected after IPTG induction.
5. The method for prokaryotic expression of the antimicrobial peptide cecropin-BM according to claim 4, wherein said method further comprises: treating the centrifugally crushed cell bacterium liquid by using PBS (phosphate buffer solution) heavy suspension containing urea and imidazole, centrifuging and collecting precipitates to obtain supernatant;
and purifying the supernatant by adopting a Ni column affinity chromatography to obtain the purified antibacterial peptide cecropin-BM.
6. Use of the antimicrobial peptide cecropin-BM according to claim 3 for the preparation of a preparation for inhibiting the growth of Escherichia coli and Staphylococcus aureus.
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