CN113234137A - Application of CXCL20a protein isolated from grass carp as antibacterial peptide - Google Patents
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Abstract
The invention belongs to the field of biotechnology, and particularly relates to application of CXCL20a protein separated from grass carp as antibacterial peptide, wherein the amino acid sequence of the protein is shown as SEQ ID NO:1, the CXCL20a has high-efficiency antibacterial effect and can play a role in drug-resistant escherichia coli, drug-resistant staphylococcus aureus, escherichia coli, staphylococcus aureus, streptococcus agalactiae, micrococcus luteus, pseudomonas fluorescens, yersinia, vibrio fluvialis and aeromonas hydrophila; compared with other antibacterial peptides, CXCL20a still has good antibacterial activity under high temperature (121 ℃) and high pressure (0.12 MPa). The special fish CXCL20a has good antibacterial activity and high temperature and pressure resistance, can be used for preparing antibacterial biological products and disinfectants, can overcome adverse effects caused by abuse of antibiotics, and has good application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of CXCL20a protein separated from grass carp as antibacterial peptide.
Background
Antibiotics the major weapons of humans against bacterial infectious diseases have gained some success in the past decades for the treatment of bacterial infectious diseases, but in recent years, especially in the aquaculture industry, with the abuse of antibiotics, more and more pathogenic microorganisms have developed resistance to traditional antibiotics, and the emergence of "superbacteria" has created almost all antibiotics, and has created a great threat to human health and the development of the aquaculture industry. Therefore, the search for new drugs that are free from bacterial resistance and can replace antibiotics is urgent.
Chemokines are a class of secreted proteins encoded by 70-100 amino acids with 6-14kDa molecular weight, with the primary function of regulating the directed migration of a wide variety of immune cells under inflammatory and normal physiological conditions. The number of fish chemokines varies depending on the fish species, the main subtypes are CC, CXC and XC, the grass carp CXCL20a belongs to CXC subtype family proteins, and CXCL20a is a fish-specific protein for which no relevant functional studies are found, and only the transcriptional level is retained.
Disclosure of Invention
The invention aims to provide application of CXCL20a protein separated from grass carp as antibacterial peptide, wherein CX CL20a protein is shown in SEQ ID No. 1.
In order to achieve the purpose, the invention adopts the following technical measures:
the CXCL20a protein separated from grass carp is used as antibacterial peptide, the CXCL20a protein is shown in SEQ ID NO.1, and the polynucleotide for coding the CXCL20a protein is preferably SEQ ID NO.2 or SEQ ID NO. 3.
In the above applications, the bacteria with antibacterial peptide action include but are not limited to: drug-resistant Escherichia coli, drug-resistant Staphylococcus aureus, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Micrococcus luteus, Pseudomonas fluorescens, Yersinia, Vibrio fluvialis, and Aeromonas hydrophila.
In the application, the preferable CXCL20a protein is obtained by a eukaryotic expression mode, and the method comprises the steps of carrying out codon optimization on a polynucleotide for coding CXCL20a protein according to pichia pastoris preferred codons, wherein the optimized gene segment is shown as SEQ ID NO.3, directionally connecting the gene segment with a pPIC9k expression vector, carrying out enzyme digestion linearization through Sal I, then electrically transferring into host pichia pastoris for expression, and extracting recombinant protein through cation exchange chromatography.
Compared with the prior art, the invention has the following advantages:
the applicant firstly discovers that the grass carp CXCL20a has a direct bactericidal function similar to that of antibacterial peptide, and compared with the popular antibacterial peptide, the CXCL20a is resistant to high temperature and high pressure and still has good antibacterial activity under the condition.
The method is characterized in that the protein CXCL20a is expressed in a eukaryotic expression mode, the operation is simple and rapid, the time requirement is short, the expression yield is high, the method is suitable for industrialization, and the method has a good application value. For example, as a medicament for the treatment of bacterial diseases.
Drawings
FIG. 1 shows the construction of pPIC9k recombinant expression vector for CXCL20a gene.
Fig. 2 shows the results of screening CXCL20a high-expression strains using G418.
Fig. 3 is the result of detecting CXCL20 a-positive transformants by PCR.
Fig. 4 shows the results of CXCL20a expression and methanol concentration optimization.
Fig. 5 shows the results of CXCL20a protein purification.
Detailed Description
The preparation and use of CXCL20a according to the present invention are specifically described below by way of examples. These examples are merely illustrative of the invention and are not intended to be limiting. The technical solutions of the present invention, unless otherwise specified, are conventional in the art, and reference is made to "microbiology experiments" (fourth edition). The grass carp CXCL20a protein used in the embodiment of the invention is obtained by a eukaryotic expression mode by utilizing a conventional mode in the field. For example, other protein expression methods or CXCL20a protein obtained by direct synthesis can achieve the technical effects of the present invention.
Example 1:
construction of pPIC9k recombinant expression System containing CXCL20a Gene
According to pichia pastoris preferred codons, a gene synthesis method is utilized to optimize grass carp CXCL20a nucleotide sequence, two enzyme cutting sites of SnaB I and EcoR I are respectively added at two ends, the two enzyme cutting sites are directly inserted behind an alpha-secretory signal peptide in pPIC9k, and a TAA terminator is inserted behind a target nucleotide sequence to terminate translation. Then, carrying out enzyme digestion linearization by using Sal I, and electrically transferring into host pichia pastoris competent cells by adopting an electrical transformation method. And (3) screening the expression strains through G418 to obtain strains capable of expressing the recombinant fusion protein.
The method comprises the following specific steps:
(1) according to the codon preference of pichia pastoris, the grass carp CXCL20a nucleotide sequence is subjected to codon optimization, the optimized sequence is inserted into pPIC9k behind an alpha-secretion signal peptide, and a TAA terminator is inserted into the target nucleotide sequence when the codon is optimized so as to ensure the translation termination of the target protein (figure 1). The nucleotide and amino acid sequences are shown in a sequence table 1:
TABLE 1 grass carp CXCL20a nucleotide and amino acid sequence
(2) And (4) linearizing the target sequence. The Sal I enzyme digestion linearization reaction system is shown in the table:
TABLE 2 linearization reaction systems
| Components | Dosage form |
| cDNA(500ng/μL) | 30μl |
| Sal I enzyme | 5μl |
| Sal I enzyme Buffer | 5μl |
| H2O | 10μl |
| Total volume | 50μl |
Adding into a 1.5ml EP tube according to a linear reaction system, placing into a 37 ℃ water bath kettle, performing enzyme digestion for 30min, and then recovering.
(3) Yeast competent cells were prepared. Picking up a yeast single colony, inoculating the yeast single colony into a 50ml triangular flask containing 5ml of YPD culture medium, and culturing overnight at 30 ℃ and 300 r/min; inoculating 100-; centrifuging the cell culture at 4 deg.C and rotation speed of 1500g for 5min, and resuspending the thallus precipitate with 50ml of ice-precooled sterile water; centrifuging under the same conditions, and resuspending the thallus precipitate with 25ml of ice-precooled sterile water; centrifuging under the same conditions, and resuspending the thallus precipitate with 20ml of ice-precooled 1mol sorbitol solution; centrifugation was carried out under the same conditions, and the pellet was resuspended in 1ml of ice-cold sorbitol solution, the total volume of which was about 1.5 ml.
(4) The linearized plasmid was electroporated into yeast competent cells. And (3) uniformly mixing the linearized plasmid obtained in the step (2) with 100 mu l of the yeast competent cells obtained in the step (3), transferring the mixture into an ice-precooled electric transformation cup (ice bath for 5min), shocking by using a voltage of 1.5kv, a resistance of 200 omega and a capacitance of 25 mu F, immediately adding 1ml of ice-precooled sorbic acid solution to uniformly mix the thalli after the electric shock is finished, transferring the electric transformation solution into a 1.5ml EP tube in a super clean bench, standing for 1h at 30 ℃, then separating for 5min at 1500g, sucking 500 mu l of supernatant, adding 500 mu l of YPD culture medium, shaking for 30min at 30 ℃ at 250r, centrifuging again, sucking 600 mu l of supernatant, and re-suspending the thalli. 200. mu.l of the cell suspension was spread on YPD plates, and the plates were incubated at 30 ℃ for 2-4 days until single colonies appeared.
(5) G418 screening expression strains. The single colonies grown in YPD were inoculated by photolithography onto G418 plates (0.25mg/ml, 0.5mg/ml, 1.0mg/ml) of different concentrations (FIG. 2), and the dominant positive strains on the same plates were subjected to PC R assay (FIG. 3) under the following conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 60s, 30 cycles, and final extension at 72 ℃ for 10 min. The reaction system is shown in table 3:
TABLE 3 CXCL20a PCR assay System
The strains tested for correctness were then induced to obtain expression strains.
(6) The expression strain is induced and the methanol induction concentration is optimized. And (3) performing amplification culture on the monoclonal colony selected in the step (5) by using a BM MY culture medium, centrifuging for 5min at 4 ℃ under 1500g, transferring into a BMGY culture medium for induction culture for 5d, performing target protein detection by using fermentation supernatant, and optimizing the induction concentration of pure methanol to determine that the induction concentration of the pure methanol is 1.0% (figure 4).
Example 2:
purification of CXCL20a recombinant proteins
The fermentation supernatant of the scale-up induction was collected and CXCL20a was purified using cations according to the nature of the protein.
The method comprises the following specific steps:
the fermentation supernatant was incubated with cationic filler overnight at 4 ℃. The solution was then passed through a cation exchange chromatography column at a flow rate of 0.5mL/min, after which the heteroproteins were eluted at a flow rate of 1mL/min with 20mmol/L Tris, 150mmol/L NaCl, pH 8.0 buffer. Finally, the target protein is eluted by using a flow rate gradient of 20mmol/L Tris, 150-. Finally, 80. mu.l of the purified protein solution was aspirated, 20. mu.l of 5 XSDS loading buffer was added, and detection was performed by SDS-PAGE (FIG. 5). As can be seen from FIG. 5, CXCL20a protein was successfully purified by the above method, and the purified protein was pure and the concentration of the purified protein reached 2 mg/ml.
Example 3:
antimicrobial Activity detection
Testing bacteria: vibrio fluvialis (ATCC 33847), Aeromonas hydrophila (Aeromonas hydrophila, ATCC7966), Streptococcus agalactiae (Streptococcus agalactiae, ATCC13813), Staphylococcus aureus (Staphylococcus aureus, ATCC25923), Escherichia coli (Escherichia coli, ATCC25922), Micrococcus luteus (Micrococcus luteus, ATCC 10240))Pseudomonas fluorescens (AT CC17397), Yersinia (Yersiniavan Loghem, DSM 18506).
The method comprises the following specific steps:
(1) eight bacteria stored at-80 ℃ were: vibrio fluvialis, Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus, Streptococcus agalactiae, Micrococcus luteus, Pseudomonas fluorescens and Yersinia are dissolved at 4 ℃, taken out and balanced to room temperature, then inoculated with a small amount of broth liquid culture medium respectively, and cultured in a constant temperature shaking table at 28 or 37 ℃ for 18 hours to logarithmic phase according to the bacterial characteristics.
(2) Inoculating the activated bacteria liquid to broth agar solid slant culture medium, culturing at 28 or 37 deg.C for 18 hr, washing thallus Porphyrae with sterile water, and diluting to 5 × 106CFU/ml。
(3) And uniformly mixing 50 mu l of diluted bacterial liquid with 50 mu l of CXCL20a solution with different concentrations, putting the mixture into a constant-temperature water bath kettle at 28 ℃ for incubation for 3 hours, taking out 50 mu l of mixed liquid to coat on a flat plate, performing 3 parallel operations on each group, performing inverted culture at 28 or 37 ℃ for 12 hours, counting bacterial colonies and taking a picture, wherein the negative control is bacterial liquid and sterile water.
(4) The bacteriostatic rate was calculated according to the following formula:
the bacteriostasis rate (100 percent) is (number of negative control colonies-number of experimental group colonies)/number of negative control colonies multiplied by 100 percent
(5) And (4) analyzing results:
TABLE 4 inhibition of CXCL20a against eight bacteria
As can be seen from Table 4, CXCL20a has very high inhibition rates on 8 bacteria of Vibrio fluvialis, Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus, Streptococcus agalactiae, Micrococcus luteus, Pseudomonas fluorescens and Yersinia at low concentrations, indicating that CXCL20a has obvious inhibition effects on the 8 bacteria. From the inhibition rates shown in the table, CXCL20a has a very strong inhibitory effect on staphylococcus aureus, pseudomonas fluorescens and streptococcus agalactiae.
Example 4:
detection of anti-multidrug-resistant bacterial activity
Testing bacteria: methicillin-resistant Staphylococcus aureus (A TCC43300), beta-lactamase-resistant antibiotic E.coli (Escherichia coli (Migula) Castellani and C halomers, ATCC35218), pathogenic multi-resistant E.coli PCN033(Genome analysis and in vivo vitamin understanding of pathogenic antibiotic strain PCN 033).
The method comprises the following specific steps:
(1) firstly, 3 kinds of bacteria preserved at-80 ℃ are: dissolving methicillin-resistant staphylococcus aureus, beta-lactamase-resistant antibiotic escherichia coli and pathogenic multi-drug-resistant escherichia coli PCN033 at 4 ℃, taking out and balancing to room temperature, then respectively inoculating and activating a small amount of broth liquid culture medium, and carrying out constant temperature shaking table culture at 37 ℃ for 18 hours to logarithmic phase.
(2) Inoculating the activated bacteria liquid to a broth agar solid slant culture medium, culturing at 37 deg.C for 18 hr, and adding sterile waterWashing off thallus Porphyrae, and diluting to 5 × 106CFU/ml。
(3) And uniformly mixing 50 mu l of diluted bacterial liquid with 50 mu l of CXCL20a solution with different concentrations, putting the mixture into a constant-temperature water bath kettle at 28 ℃ for incubation for 3 hours, taking out 50 mu l of mixed liquid to coat the mixture on a flat plate, performing inverted culture at 37 ℃ for 12 hours, counting bacterial colonies and taking a picture, wherein the negative control is bacterial liquid and sterile water.
(4) The bacteriostatic rate was calculated according to the following formula:
the bacteriostasis rate (100 percent) is (number of negative control colonies-number of experimental group colonies)/number of negative control colonies multiplied by 100 percent
(5) And (4) analyzing results:
TABLE 5 inhibition of CXCL20a against three bacteria
As can be seen from Table 5, CXCL20a has a very high inhibition rate at low concentrations against multi-drug resistant strains such as methicillin-resistant Staphylococcus aureus, beta-lactamase-resistant antibiotic Escherichia coli, pathogenic multi-drug resistant Escherichia coli PCN 0333, indicating that CXCL20a has an obvious inhibition effect on these 3 bacteria.
Example 5:
detection of antibacterial Activity under high temperature and high pressure test bacteria: escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923).
The method comprises the following specific steps:
(1) 100 μ l of CXCL20a protein solution was filled in a 1.5ml EP tube, and heated and pressurized in an autoclave (temperature 121 ℃ C., high pressure 0.12MPa) for 30 min.
(2) Selecting a gram-negative bacterium (Escherichia coli) and a gram-positive bacterium (Staphylococcus aureus) to carry out inoculation and activation on a small amount of broth liquid culture medium, and respectively carrying out shake cultivation at a constant temperature of 37 ℃ for 18 hours to logarithmic phase.
(3) Inoculating the activated bacterial liquid to a broth agar solid slant culture medium, culturing at 37 deg.C for 18 hr, and washing with sterile waterDiluting to 5X 106CFU/ml。
(4) And uniformly mixing 50 mu l of diluted bacterial liquid with 50 mu l of CXCL20a solution with different concentrations, putting the mixture into a constant-temperature water bath kettle at 28 ℃ for incubation for 3 hours, taking out 50 mu l of mixed liquid to coat on a flat plate, performing 3 parallel operations on each group, performing inverted culture at 28 or 37 ℃ for 12 hours, counting bacterial colonies and taking a picture, wherein the negative control is bacterial liquid and sterile water.
(5) The bacteriostatic rate was calculated according to the following formula:
the bacteriostasis rate (100 percent) is (number of negative control colonies-number of experimental group colonies)/number of negative control colonies multiplied by 100 percent
(6) And (4) analyzing results:
TABLE 6 inhibition of CXCL20a against two bacteria at high temperature and high pressure
As can be seen from table 6, CXCL20a still has good antibacterial activity against gram-negative bacteria (e.coli) and gram-positive bacteria (staphylococcus aureus) under high temperature and high pressure. The CXCL20a can resist high temperature and high pressure, has obvious inhibition effect on gram negative and positive bacteria and provides possibility for preparing biological products.
Sequence listing
<110> university of agriculture in Huazhong
<120> use of CXCL20a protein isolated from grass carp as antibacterial peptide
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Met Arg Lys Ala Met Lys Pro Lys Tyr Ile His Ala Ala Glu Leu Phe
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Pro Pro Ser Ala Ser Cys Ser Lys Thr Glu Ile Ile Leu Thr Leu Asn
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Arg Lys Gly Lys Gly Lys Gly Arg Gly Lys Gly Leu Lys Val Cys Leu
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Asp Pro Tyr Glu Lys Gln Gly Arg Arg Leu Leu Lys Ser Lys Gly Ile
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gggcatggag cagggatttc acagcgctgt ttatgcagag gcagaatgcg gaaagcaatg 60
aagcctaaat acattcatgc agctgaattg ttcccaccaa gtgcctcctg ctcaaagacc 120
gagattatac tgactctcaa tagaaaagga aagggaaaag gaaggggaaa ggggttaaag 180
gtgtgtctgg atccatatga gaagcaagga cggagattac tgaagagcaa agggattcaa 240
aacaaaaagc aaaagaacag aggaaggaaa ggaaggaata aaaagtctga tgtcaaggat 300
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ggtcatggtg ctggaatctc tcagaggtgc ttgtgcagag gaaggatgag gaaggccatg 60
aagccaaagt acatccacgc tgccgagttg ttcccaccat ctgcctcttg ctctaagacc 120
gagatcatct tgacccttaa tagaaaggga aagggtaagg gaaggggaaa gggattgaag 180
gtctgcttgg acccatacga gaagcaagga aggaggcttt tgaagtctaa gggtattcaa 240
aacaagaagc agaagaatag aggtaggaag ggaaggaaca agaaatctga tgttaaagat 300
gattga 306
Claims (6)
1. The CXCL20a protein separated from grass carp is used for preparing antibacterial peptide, and the CXCL20a protein is shown in SEQ ID NO. 1.
2. The application of the polynucleotide shown in SEQ ID NO.1 in preparing antibacterial peptide.
3. The use of claim 2, wherein the polynucleotide is represented by SEQ ID No.2 or SEQ ID No. 3.
4. The use according to claim 1 or claim 2, wherein the antibacterial peptide is selected from the group consisting of: drug-resistant Escherichia coli, drug-resistant Staphylococcus aureus, Escherichia coli: (Escherichia coli) Staphylococcus aureus (1)Staphylococcus aureaus) Streptococcus agalactiae (1)Streptococcus agalactiae) Micrococcus luteus (a)Micrococcus luteus) Pseudomonas fluorescens (A)Pseudomonas fluorescens) Yersinia, Yersinia: (Yersiniavan Loghem) Vibrio fluvialis (Vibrio fluvialis)Vibrio fluvialis) And/or Aeromonas hydrophila ()Aeromonas hydrophila）。
5. The use of claim 1, wherein the CXCL20a protein is obtained by eukaryotic expression, comprising the step of directionally linking the gene fragment shown in SEQ ID NO.3 to the pPIC9k expression vectorSalI carry outAfter enzyme digestion linearization, the recombinant protein is expressed after being electrically transferred into host pichia pastoris and is extracted by cation exchange chromatography.
6. The use of claim 4, wherein the drug-resistant E.coli is beta-lactamase-resistant antibiotic E.coli (E.coli)Escherichia coli (Migula) Castellani and Chalmers) Or pathogenic multi-drug resistant Escherichia coli PCN033, wherein the drug resistant Staphylococcus aureus is methicillin resistant Staphylococcus aureus(s) ((R))Methicillin-resistant Staphylococcus aureus）。
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