A recombinant chicken antibacterial peptide against drug-resistant staphylococcus aureus

Abstract

The application provides a recombinant chicken antibacterial peptide against drug-resistant Staphylococcus aureus and belongs to the field of veterinary technology. The recombinant chicken antibacterial peptide is expressed by genetically engineering recombinant Escherichia coli. Three active regions of natural chicken antibacterial peptides are fused, and two disulfide bonds are introduced to stabilize the structure of the recombinant chicken antibacterial peptide and enhance the affinity of the recombinant chicken antibacterial peptide to the outer membrane protein of Staphylococcus aureus. The minimum inhibitory concentration of the recombinant chicken antibacterial peptide to drug-resistant Staphylococcus aureus is significantly lower than that of the three natural chicken antibacterial peptides, and the minimum inhibitory concentration of 8 μg / mL can completely inhibit the formation of a biological membrane of a drug-resistant Staphylococcus aureus USA300 strain. The recombinant chicken antibacterial peptide has good antibacterial effect on different drug-resistant Staphylococcus aureus isolated from a chicken farm, and can effectively solve the problem of drug-resistant Staphylococcus aureus infection in the chicken farm.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary technology, and particularly relates to a recombinant chicken antibacterial peptide against drug-resistant Staphylococcus aureus. BACKGROUND

[0002] With the development of broiler breeding technology, the breeding of fast-growing broiler varieties, the shortening of the breeding cycle of broilers, and the increase in the weight of broiler ketones, the legs of broilers are prone to joint problems due to the fast growth and large body weight, and the joint disease of broilers brings great harm to the broiler breeding industry. Among them, the most common chicken staphylococcal joint disease is a bacterial infectious disease caused by Staphylococcus aureus, and the main characteristics are joint swelling, septic arthritis and lameness, which are mainly seen in chicks and broilers. Staphylococcus aureus is a gram-positive coccus that widely exists in the environment. Pathogenic strains often produce toxins (such as enterotoxin and hemolysin) and enzymes (such as coagulase), leading to tissue necrosis and suppuration. Acute infection of broilers causes sudden lameness, tarsal joint and toe joint swelling, and a hot and painful feeling when palpated. The affected chickens are depressed, and have reduced appetite, and severe cases are bedridden. Sepsis may occur, and the mortality rate increases. The joints of the affected chickens are swollen and deformed, and contain purulent or caseous exudates. Long-term lameness, growth retardation and gradual weight loss occur. This causes great economic losses to the broiler breeding industry.

[0003] Due to high breeding density, chicken staphylococcal joint disease is prone to occur, and the effect of vaccine immunization is not ideal. Antibiotics are commonly used to treat Staphylococcus aureus. However, the problem of drug resistance of Staphylococcus aureus is becoming more and more serious due to long-term use of antibiotics in veterinary clinics. Antibiotic treatment is often ineffective, and brings more serious problems of antibiotic abuse. Antimicrobial peptides (AMPs) are a class of small molecule polypeptides with broad-spectrum antibacterial activity naturally existing in organisms, and chicken antimicrobial peptides play a key role in immune defense, especially in resisting pathogenic microbial infection. Chicken antimicrobial peptides are an important part of the natural immune system, have broad-spectrum antibacterial and immunomodulatory functions, and the biggest advantage of chicken antimicrobial peptides is that they rarely develop drug resistance, and have broad prospects in the fields of healthy breeding and medicine. Current research focuses on improving the stability, activity and large-scale production of chicken antimicrobial peptides to promote practical application. SUMMARY

[0004] The present application belongs to the field of veterinary technology, and particularly relates to a recombinant chicken antibacterial peptide against drug-resistant Staphylococcus aureus.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] Firstly, the present application provides a recombinant chicken antibacterial peptide against drug-resistant Staphylococcus aureus, and the amino acid sequence of the recombinant chicken antibacterial peptide is SEQ ID NO. 1.

[0007] Further, the nucleotide sequence of the recombinant chicken antibacterial peptide is SEQ ID NO. 2.

[0008] Further, the preparation method of the recombinant chicken antibacterial peptide comprises the following steps:

[0009] (1) picking E. coli BL21 / pET28a-rCathelicidin single colony and inoculating in 10 ml LB culture solution containing 50 μg / ml kanamycin, 37℃, 220 r / min oscillation culture for 16 hours;

[0010] (2) transferring the culture solution containing 50 μg / ml according to 1% amount, 37℃, 220 r / min oscillation culture until OD600nm is 0.6-0.8, adding 0.5 mol / L α-lactose to the final concentration of 0.03 mol / L, 37℃, and culturing for 6 hours again;

[0011] (3) centrifugal collection of bacterial body, adding 10 times volume of PBS to bacterial body suspension, ultrasonic crushing, centrifugal collection of protein supernatant;

[0012] (4) the supernatant is filtered through 0.22 μm filter, purified by nickel column, and the purified protein is stored at -20℃ for standby.

[0013] Beneficial effects: the application analyzes the structure of three kinds of natural chicken antibacterial peptides, fuses the active regions of the three kinds of natural chicken antibacterial peptides, introduces two disulfide bonds to stabilize the structure of the recombinant chicken antibacterial peptide, and enhances the affinity to the outer membrane protein of Staphylococcus aureus. The minimum inhibitory concentration of the recombinant chicken antibacterial peptide to drug-resistant Staphylococcus aureus is significantly lower than that of the three kinds of natural chicken antibacterial peptides, and the minimum inhibitory concentration of 8 μg / mL can completely inhibit the formation of the biofilm of the drug-resistant Staphylococcus aureus USA300 strain. The recombinant chicken antibacterial peptide has good antibacterial effect on different drug-resistant Staphylococcus aureus isolated from a chicken farm, and can effectively solve the problem of drug-resistant Staphylococcus aureus infection in the chicken farm. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 SDS-PAGE electrophoretogram of recombinant chicken antibacterial peptide protein;

[0015] Figure 2 Minimum inhibitory concentration determination diagram of chicken antibacterial peptide;

[0016] Figure 3 Inhibition effect detection diagram of recombinant chicken antibacterial peptide on the formation of Staphylococcus aureus biofilm;

[0017] Figure 4 Antibacterial effect detection diagram of recombinant chicken antibacterial peptide on different drug-resistant Staphylococcus aureus in a chicken farm. DETAILED DESCRIPTION

[0018] Example 1: Design of recombinant chicken antibacterial peptide sequence

[0019] (1) Retrieval of chicken antibacterial peptide amino acid sequence The chicken antibacterial peptide amino acid sequence was retrieved from GenBank, with cathelicidin-1 having the accession number AMY26515.1, cathelicidin-2 having the accession number AMY26516.1, and cathelicidin-3 having the accession number AMY26517.1.

[0020] (2) Design and analysis of recombinant chicken antibacterial peptide The structures of cathelicidin-1, cathelicidin-2, and cathelicidin-3 were analyzed, and the active regions of the three natural chicken antibacterial peptides were fused. Two disulfide bonds were introduced to stabilize the structure of the recombinant chicken antibacterial peptide and enhance its affinity for Staphylococcus aureus outer membrane protein. The designed recombinant chicken antibacterial peptide amino acid sequence is SEQ ID NO. 1.

[0021] Example 2: Expression of recombinant chicken antibacterial peptide protein

[0022] (1) Synthesis of recombinant chicken antibacterial peptide gene According to the recombinant chicken antibacterial peptide amino acid sequence of SEQ ID NO. 1, the codons of Escherichia coli were optimized, and NdeI and NotI restriction enzyme sites were added to the upstream and downstream, respectively. The gene synthesis company was commissioned to synthesize SEQ ID NO. 3, and it was cloned into the pUC57 plasmid, named pUC57-rCathelicidin.

[0023] (2) Construction of expression plasmid pUC57-rCathelicidin was double digested with NdeI and NotI restriction enzymes, and the rCathelicidin gene fragment was recovered. pET28a plasmid was double digested with NdeI and NotI restriction enzymes and recovered. pET28a and rCathelicidin gene fragments were ligated with T4 ligase to construct the expression plasmid pET28a-rCathelicidin.

[0024] (3) Construction of recombinant expression strain pET28a-rCathelicidin plasmid was transformed into E. coli BL21 competent cells, and single colonies were picked for PCR identification of positive transformants, named E. coli BL21 / pET28a-rCathelicidin.

[0025] (4) Expression and identification of the target protein Pick E. coli BL21 / pET28a-rCathelicidin single colony, inoculated in 10 ml of LB medium containing 50 μg / ml kanamycin, 37 °C, 220 r / min shaking culture 16 hours. According to the amount of 1% transfer containing 50 μg / ml of LB culture medium, 37 °C, 220 r / min shaking culture to OD600nm is 0.6~0.8. Add 0.5 mol / L of α-lactose to a final concentration of 0.03 mol / L, 25 °C, 220 r / min shaking culture 6h. After taking out 12000 r / min centrifugal 10 min, collect the bacteria. Add bacteria weight 10 times the volume of PBS for bacteria suspension, ultrasonic broken. 4 °C, 12000 r / min centrifugal 10 min, collect the protein supernatant.

[0026] (5) Purification of recombinant protein The supernatant was moved into a new tube, filtered through a 0.22 μm filter, and then added to a Ni-NTA chromatography column treated with Binding buffer, and then shaken at room temperature for 30 min. Then, the resin was completely settled at room temperature, and the liquid was naturally flowed out. Then, the chromatography column was washed with Binding buffer for 10 times the column volume, Washing buffer (8 M urea, 0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9) for 6 times the column volume, and finally Elution buffer (6 M urea, 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) for 6 times the column volume. The eluate was collected, and an appropriate amount of eluate was subjected to SDS-PAGE electrophoresis detection. The electrophoresis detection results are shown in Figure 1 . The purified protein was stored at -20 °C for standby.

[0027] Example 3: In vitro antibacterial effect determination of chicken antibacterial peptide on Staphylococcus aureus

[0028] (1) Bacterial culture and preparation of bacterial solution The drug-resistant Staphylococcus aureus USA300 strain glycerol stock was cultured at 37 °C, 200 r / min for 3 generations, and finally obtained bacteria in the logarithmic growth phase. Centrifuged at 3 000 x g for 10 min at 4 °C, discarded the supernatant, resuspended with physiological saline, and adjusted OD600 to 1.0 (about 4 x 10 8 cfu / mL), diluted and used for subsequent experiments.

[0029] (2) Minimum inhibitory concentration determination The drug-resistant Staphylococcus aureus USA300 strain 3 generation bacterial solution was diluted to 1 x 10 8The bacterial culture was diluted with PBS to concentrations of cfu / mL, and chicken antimicrobial peptides cathelicidin-1, cathelicidin-2, cathelicidin-3, and recombinant chicken antimicrobial peptide rCathelicidin were diluted to concentrations of 2, 4, 8, 16, 32, and 64 μg / mL, respectively. 100 μL of each bacterial culture and antimicrobial peptide dilution was mixed in 96-well polystyrene microplates (each concentration was repeated three times). After incubating the 96-well plates at 37°C for 24 h, the minimum inhibitory concentration (MIC) was determined by visually observing the turbidity within the wells.

[0030] Depend on Figure 2 It can be seen that the minimum inhibitory concentrations (MICs) of chicken antimicrobial peptides cathelicidin-1, cathelicidin-2, cathelicidin-3, and recombinant chicken antimicrobial peptide rCathelicidin against drug-resistant Staphylococcus aureus strain USA300 are 16 μg / mL, 32 μg / mL, 16 μg / mL, and 8 μg / mL, respectively. The recombinant chicken antimicrobial peptide rCathelicidin has the lowest MIC and the best antimicrobial effect.

[0031] Example 4: Inhibition of Staphylococcus aureus biofilm formation by recombinant chicken antimicrobial peptides

[0032] The third-generation bacterial culture of Staphylococcus aureus strain USA300 was diluted to 1×10⁻⁶. 8 CFU / mL was inoculated into 96-well plates. Subsequently, 0, 2, 4, 8, and 16 μg / mL of recombinant chicken antimicrobial peptide solution were added to each well to bring the final volume to 200 μL. The 96-well plates were incubated statically at 37°C for 24 h. After incubation, the supernatant was discarded, and each well was gently washed three times with PBS to remove unbound bacteria. Next, the plates were fixed with 10% methanol for 15 min and dried at 60°C for 2 h to stabilize the biofilm structure. Then, the plates were stained with 0.5% crystal violet for 15 min, and unbound dye was washed away with sterile PBS. Finally, the plates were destained with anhydrous ethanol, and the OD value was measured using a microplate reader with PBS as a blank control. 595 nm The absorbance was measured at the specified wavelength.

[0033] Depend on Figure 3 It is known that the recombinant chicken antimicrobial peptide rCathelicidin at a concentration of 8 μg / mL or higher has a good inhibitory effect on biofilm formation of drug-resistant Staphylococcus aureus strain USA300, and the MIC concentration can completely inhibit biofilm formation.

[0034] Example 5: Determination of the in vitro antibacterial effect of recombinant chicken antimicrobial peptides against drug-resistant strains in chicken farms

[0035] (1) Bacterial Culture and Preparation of Bacterial Fluid: Methicillin-resistant Staphylococcus aureus R1, erythromycin-resistant Staphylococcus aureus R2, clindamycin-resistant Staphylococcus aureus R3, and multidrug-resistant Staphylococcus aureus R4 isolated from a chicken farm were cryopreserved with glycerol and cultured for 3 generations at 37 °C and 200 r / min with shaking to obtain bacteria in the logarithmic growth phase. The bacteria were centrifuged at 3000 × g for 10 min at 4 °C, the supernatant was discarded, and the suspension was resuspended in physiological saline. The OD600 was adjusted to 1.0 (approximately 4 × 10⁻⁶). 8 (cfu / mL), diluted for subsequent experiments.

[0036] (2) Determination of minimum inhibitory concentration: Dilute the third-generation bacterial suspensions of the above four drug-resistant Staphylococcus aureus strains to 1×10⁻⁶. 8 The bacterial culture was diluted with PBS to concentrations of 2, 4, 8, 16, 32, and 64 μg / mL. 100 μL of each bacterial culture and the diluted recombinant chicken antimicrobial peptide solution were mixed in 96-well polystyrene microplates (each concentration was repeated three times). After incubating the 96-well plates at 37°C for 24 h, the minimum inhibitory concentration (MIC) was determined by visually observing the turbidity within the wells.

[0037] Depend on Figure 4 The results show that the minimum inhibitory concentration (MIC) of the recombinant chicken antimicrobial peptide rCathelicidin against methicillin-resistant Staphylococcus aureus (MRSA) strain R1 isolated from chicken farms is 8 μg / mL, against erythromycin-resistant Staphylococcus aureus (SRA) strain R2 is 8 μg / mL, against clindamycin-resistant Staphylococcus aureus (SRA) strain R3 is 8 μg / mL, and against multidrug-resistant Staphylococcus aureus (MDR-SRA) strain R4 is 16 μg / mL. This demonstrates that the recombinant chicken antimicrobial peptide rCathelicidin exhibits good antimicrobial effects against various drug-resistant Staphylococcus aureus strains isolated from chicken farms.

Claims

1. A recombinant chicken antimicrobial peptide targeting drug-resistant Staphylococcus aureus, characterized in that, The amino acid sequence of the recombinant chicken antimicrobial peptide is SEQ ID NO.

1.

2. A method for preparing the recombinant chicken antimicrobial peptide as described in claim 1, characterized in that, The preparation method of the recombinant chicken antimicrobial peptide includes the following steps: (1) The pET28a and rCathelicidin genes were ligated using T4 ligase to construct the expression plasmid pET28a-rCathelicidin. The pET28a-rCathelicidin plasmid was transformed into E. coli BL21 competent cells. Single colonies were picked and positive transformants were identified by PCR and named E. coli BL21 / pET28a-rCathelicidin. The nucleotide sequence of the rCathelicidin gene is SEQ ID NO.

2. (2) Pick a single colony of E. coli BL21 / pET28a-rCathelicidin and inoculate it into 10 ml of LB medium containing 50 μg / ml kanamycin. Incubate at 37°C and 220 r / min for 16 hours with shaking. (3) Transfer LB medium containing 50 μg / ml at a rate of 1%, and culture at 37℃ and 220 r / min with shaking until the OD600nm is 0.6~0.

8. Add 0.5 mol / L α-lactose to the final concentration of 0.03 mol / L, and culture at 37℃ for another 6 h. (4) Centrifuge to collect bacterial cells, add PBS with a volume of 10 times the weight of the bacterial cells to suspend the bacterial cells, sonicate to disrupt, and centrifuge to collect the protein supernatant; (5) The supernatant was filtered through a 0.22 μm filter and purified with a nickel column. The purified protein was stored at -20°C for later use.

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