Recombinant chicken antibacterial peptide aiming at drug-resistant staphylococcus aureus

Abstract

The invention provides a recombinant chicken antibacterial peptide aiming at drug-resistant staphylococcus aureus, and belongs to the technical field of veterinarians. The recombinant chicken antibacterial peptide is expressed through genetic engineering recombinant escherichia coli. Three natural chicken antibacterial peptide active regions 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 staphylococcus aureus outer membrane protein. The minimum inhibitory concentration of the recombinant chicken antibacterial peptide to the drug-resistant staphylococcus aureus is obviously lower than that of three natural chicken antibacterial peptides, and the formation of a biomembrane of a USA300 strain of the drug-resistant staphylococcus aureus can be thoroughly inhibited when the minimum inhibitory concentration is 8 mu g / mL. The recombinant chicken antibacterial peptide has a good antibacterial effect on different drug-resistant staphylococcus aureus separated from a chicken house, and can effectively solve the infection problem of the drug-resistant staphylococcus aureus in the chicken house.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary medicine, and in particular relates to a recombinant chicken antimicrobial peptide targeting drug-resistant Staphylococcus aureus. Background Art

[0002] With the advancement of broiler farming technology and the development of fast-growing broiler breeds, the broiler breeding cycle has shortened, and the carcass weight of broilers has increased. Due to their rapid growth rate and heavy weight, broilers bear heavy loads on their legs, which can easily lead to joint problems. Coupled with joint-invading pathogens, broiler joint diseases pose a significant threat to the broiler industry. Among the most common diseases, Staphylococcal arthritis of chickens, caused by Staphylococcus aureus, is a bacterial infection characterized by joint swelling, suppurative arthritis, and lameness, and is common in chicks and broilers. Staphylococcus aureus is a Gram-positive coccus widely found in the environment. Pathogenic strains often produce toxins (such as enterotoxins and hemolysins) and enzymes (such as coagulase), leading to tissue necrosis and suppuration. Acutely infected broilers develop sudden lameness, swelling of the tarsal and toe joints, and a warm, tender sensation on palpation. Affected birds exhibit depression, loss of appetite, and, in severe cases, resignation to inactivity. Septicemia may occur, leading to an increased mortality rate. In resistant chickens, joints become swollen and deformed, and contain purulent or cheesy exudates. Long-term lameness, slow growth, and gradual weight loss cause huge economic losses to the broiler farming industry.

[0003] Due to high stocking densities, Staphylococcal arthritis is common in chickens, and vaccination is ineffective. Antibiotics are a common treatment for Staphylococcus aureus. However, the long-term use of antibiotics in veterinary clinics has led to the increasing prevalence of drug resistance in Staphylococcus aureus. Antibiotic treatment is often ineffective and leads to the serious problem of antibiotic overuse. Antimicrobial peptides (AMPs) are naturally occurring small peptides with broad-spectrum antimicrobial activity. AMPs in chickens play a key role in immune defense, particularly in combating pathogenic microbial infections. Chicken AMPs are an important component of the innate immune system, possessing broad-spectrum antimicrobial and immunomodulatory properties. Their greatest advantage is their low likelihood of developing drug resistance, holding them a promising future in both animal husbandry and medicine. Current research focuses on improving their stability, activity, and scalable production to promote their practical application. Summary of the Invention

[0004] The purpose of the present invention is to provide a recombinant chicken antimicrobial peptide targeting drug-resistant Staphylococcus aureus, belonging to the field of veterinary technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: First, the present invention provides a recombinant chicken antimicrobial peptide against drug-resistant Staphylococcus aureus, wherein the amino acid sequence of the recombinant chicken antimicrobial peptide is SEQ ID NO.1.

[0006] Furthermore, the nucleotide sequence of the recombinant chicken antimicrobial peptide is SEQ ID NO.2.

[0007] Furthermore, the preparation method of the recombinant chicken antimicrobial peptide comprises the following steps: (1) Pick a single colony of E. coli BL21 / pET28a-rCathelicidin and inoculate it into 10 ml of LB culture medium containing 50 μg / ml kanamycin. Incubate at 37°C and 220 rpm for 16 hours. (2) Transfer 1% of the culture medium to LB culture medium containing 50 μg / ml, culture at 37°C and 220 r / min with shaking until the OD600nm is 0.6-0.8, add 0.5 mol / L α-lactose to a final concentration of 0.03 mol / L, and culture at 37°C for another 6 h; (3) Collect the cells by centrifugation, add PBS (10 times the cell weight) to suspend the cells, disrupt them by ultrasonic wave, and collect the protein supernatant by centrifugation; (4) The supernatant was filtered through a 0.22 μm filter and purified using a nickel column. The purified protein was stored at -20°C until use.

[0008] Beneficial Effects: The present invention analyzes the structures of three natural chicken antimicrobial peptides, fuses the active regions of the three natural chicken antimicrobial peptides, introduces two disulfide bonds to stabilize the structure of the recombinant chicken antimicrobial peptide, and enhances its affinity for the outer membrane protein of Staphylococcus aureus. The minimum inhibitory concentration of the recombinant chicken antimicrobial peptide against drug-resistant Staphylococcus aureus is significantly lower than that of the three natural chicken antimicrobial peptides. A minimum inhibitory concentration of 8 μg / mL can completely inhibit the formation of biofilms of drug-resistant Staphylococcus aureus USA300 strain. The recombinant chicken antimicrobial peptide has a good antibacterial effect against different drug-resistant Staphylococcus aureus isolated from chicken farms, and can effectively solve the problem of drug-resistant Staphylococcus aureus infection in chicken farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 SDS-PAGE electrophoresis of recombinant chicken antimicrobial peptide protein; Figure 2 Minimum inhibitory concentration of chicken antimicrobial peptides determination chart; Figure 3 Detection of the inhibitory effect of recombinant chicken antimicrobial peptide on Staphylococcus aureus biofilm formation; Figure 4 Detection of the antibacterial effect of recombinant chicken antimicrobial peptide on different drug-resistant Staphylococcus aureus in chicken farms. DETAILED DESCRIPTION

[0010] Example 1: Design of recombinant chicken antimicrobial peptide sequences (1) Retrieval of amino acid sequences of chicken antimicrobial peptides The amino acid sequences of chicken antimicrobial peptides were retrieved from GenBank. The accession numbers of cathelicidin-1, cathelicidin-2, and cathelicidin-3 were AMY26517.1.

[0011] (2) Design of recombinant chicken antimicrobial peptides: The structures of three natural chicken antimicrobial peptides, cathelicidin-1, cathelicidin-2, and cathelicidin-3, were analyzed. The active regions of the three natural chicken antimicrobial peptides were fused together, and two disulfide bonds were introduced to stabilize the structure of the recombinant chicken antimicrobial peptides and enhance their affinity for Staphylococcus aureus outer membrane protein. The amino acid sequence of the designed recombinant chicken antimicrobial peptide is SEQ ID NO. 1.

[0012] Example 2: Expression of recombinant chicken antimicrobial peptide protein (1) Synthesis of recombinant chicken cathelicidin gene Based on the amino acid sequence of recombinant chicken cathelicidin of SEQ ID NO.1, the gene was optimized according to the codon preference of Escherichia coli, and NdeI and NotI restriction endonuclease sites were added upstream and downstream respectively. A gene synthesis company was commissioned to synthesize SEQ ID NO.3 and cloned into the pUC57 plasmid, named pUC57-rCathelicidin.

[0013] (2) Construction of expression plasmid: pUC57-rCathelicidin was double-digested with NdeI and NotI restriction endonucleases, and the rCathelicidin gene fragment was recovered. The pET28a plasmid was double-digested with NdeI and NotI restriction endonucleases and recovered. The pET28a and rCathelicidin gene fragments were ligated with T4 ligase to construct the expression plasmid pET28a-rCathelicidin.

[0014] (3) Construction of recombinant expression strain The pET28a-rCathelicidin plasmid was transformed into E. coli BL21 competent cells, and single colonies were picked for PCR identification of positive transformants, which were named E. coli BL21 / pET28a-rCathelicidin.

[0015] (4) Expression and identification of target protein A single colony of E. coli BL21 / pET28a-rCathelicidin was picked and inoculated into 10 ml of LB culture medium containing 50 μg / ml kanamycin. The culture was shaken at 37°C and 220 r / min for 16 hours. A 1% amount of the colony was transferred to LB culture medium containing 50 μg / ml kanamycin. The culture was shaken at 37°C and 220 r / min until the OD600nm was 0.6-0.8. 0.5 mol / L α-lactose was added to a final concentration of 0.03 mol / L. The culture was shaken at 25°C and 220 r / min for 6 hours. After removal, the cells were centrifuged at 12000 r / min for 10 minutes to collect the cells. PBS with a volume of 10 times the cell weight was added to suspend the cells and ultrasonically disrupted. The cells were centrifuged at 12000 r / min for 10 minutes at 4°C to collect the protein supernatant.

[0016] (5) Purification of recombinant protein: The supernatant was transferred to a new tube, filtered through a 0.22 μm filter, and then added to a Ni-NTA chromatography column treated with Binding buffer. The column was shaken at low speed at room temperature for 30 minutes, and then allowed to stand at room temperature until the resin was completely settled, and the liquid was allowed to flow out naturally. The column was then washed thoroughly with 10 column volumes of Binding buffer, 6 column volumes of Washing buffer (8 M urea, 0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9), and finally the target protein was eluted with 6 column volumes of Elution buffer (6 M urea, 500 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The eluate was collected and an appropriate amount of the eluate was used for SDS-PAGE electrophoresis detection. The electrophoresis detection results are shown in FIG. Figure 1 The purified protein was stored at -20°C until use.

[0017] Example 3: Determination of the in vitro antibacterial effect of chicken antimicrobial peptides against Staphylococcus aureus (1) Bacterial culture and preparation of bacterial solution: Glycerol-frozen resistant Staphylococcus aureus USA300 strain was cultured at 37°C and 200 rpm for three generations, and finally bacteria in the logarithmic growth phase were obtained. The culture was centrifuged at 3000×g for 10 min at 4°C, the supernatant was discarded, and the suspension was resuspended in physiological saline and the OD600 was adjusted to 1.0 (approximately 4×10 8 cfu / mL) and were used in subsequent experiments after dilution.

[0018] (2) Determination of minimum inhibitory concentration: The third generation bacterial solution of drug-resistant Staphylococcus aureus USA300 was diluted to 1×10 8cfu / mL, and chicken cathelicidin-1, cathelicidin-2, cathelicidin-3, and recombinant chicken cathelicidin were diluted with PBS to 2, 4, 8, 16, 32, and 64 μg / mL, respectively. 100 μL of each bacterial suspension and cathelicidin dilution were mixed in a 96-well polystyrene microplate (each concentration was replicated three times). After incubation at 37°C for 24 hours, the minimum inhibitory concentration (MIC) was determined by visual inspection of the well turbidity.

[0019] 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 USA300 strain were 16 μg / mL, 32 μg / mL, 16 μg / mL, and 8 μg / mL, respectively. The recombinant chicken antimicrobial peptide rCathelicidin had the lowest MIC and the best antibacterial effect.

[0020] Example 4: Inhibition of Staphylococcus aureus Biofilm Formation by Recombinant Chicken Antimicrobial Peptides The third generation bacterial solution of Staphylococcus aureus USA300 was diluted to 1×10 8 cfu / mL and inoculated into a 96-well plate. Subsequently, 0, 2, 4, 8, and 16 μg / mL of recombinant chicken antimicrobial peptide solution were added to each well to make a final volume of 200 μL. The 96-well plate was incubated at 37°C for 24 h. After the incubation, the supernatant was discarded and each well was gently washed 3 times with PBS to remove unbound bacteria. Next, it was fixed with 10% methanol for 15 min and dried at 60°C for 2 h to stabilize the biofilm structure. Subsequently, it was stained with 0.5% crystal violet for 15 min and unbound dye was washed away with sterile PBS. Finally, it was decolorized with anhydrous ethanol and PBS was used as a blank control on a microplate reader at OD 595 nm Measure absorbance at wavelength.

[0021] Depend on Figure 3 It can be seen that the recombinant chicken antimicrobial peptide rCathelicidin has a good inhibitory effect on the biofilm formation of drug-resistant Staphylococcus aureus USA300 strain when the concentration is above 8 μg / mL, and the MIC concentration can completely inhibit the formation of biofilm.

[0022] Example 5: Determination of the in vitro antibacterial effect of recombinant chicken antimicrobial peptides against drug-resistant strains in chicken farms (1) Bacterial culture and bacterial solution preparation Methicillin-resistant Staphylococcus aureus R1, erythromycin-resistant Staphylococcus aureus R2, clindamycin-resistant Staphylococcus aureus R3, and multidrug-resistant Staphylococcus aureus R4 isolated from chicken farms were cultured in glycerol frozen form at 37°C and 200 rpm for three generations, and finally bacteria in the logarithmic growth phase were obtained. The culture was centrifuged at 3000×g for 10 min at 4°C, the supernatant was discarded, and the suspension was resuspended in physiological saline and the OD600 was adjusted to 1.0 (approximately 4×10 8 cfu / mL) and were used in subsequent experiments after dilution.

[0023] (2) Minimum inhibitory concentration determination: The third generation bacterial suspensions of the above four drug-resistant Staphylococcus aureus were diluted to 1×10 8 cfu / mL, and recombinant chicken antimicrobial peptide was diluted with PBS to 2, 4, 8, 16, 32, and 64 μg / mL. 100 μL of each bacterial suspension and recombinant chicken antimicrobial peptide dilution were mixed in a 96-well polystyrene microplate (each concentration was replicated three times). After incubating the 96-well plate at 37°C for 24 hours, the minimum inhibitory concentration (MIC) was determined by visual inspection of the well turbidity.

[0024] Depend on Figure 4 The results show that the recombinant chicken antimicrobial peptide rCathelicidin has a minimum inhibitory concentration (MIC) of 8 μg / mL against methicillin-resistant Staphylococcus aureus R1 strain isolated from chicken farms, 8 μg / mL against erythromycin-resistant Staphylococcus aureus R2 strain, 8 μg / mL against clindamycin-resistant Staphylococcus aureus R3 strain, and 16 μg / mL against multidrug-resistant Staphylococcus aureus R4 strain. This indicates that the recombinant chicken antimicrobial peptide rCathelicidin has a good antibacterial effect against different drug-resistant Staphylococcus aureus strains isolated from chicken farms.

Claims

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

1.

2. The recombinant chicken antimicrobial peptide according to claim 1, characterized in that The nucleotide sequence of the recombinant chicken antimicrobial peptide is SEQ ID NO.

2.

3. The recombinant chicken antimicrobial peptide according to claim 1, characterized in that The preparation method of the recombinant chicken antimicrobial peptide comprises the following steps: (1) Pick a single colony of E. coli BL21 / pET28a-rCathelicidin and inoculate it into 10 ml of LB culture medium containing 50 μg / ml kanamycin. Incubate at 37°C and 220 rpm for 16 hours. (2) Transfer 1% of the culture medium to LB culture medium containing 50 μg / ml, culture at 37°C and 220 r / min with shaking until the OD600nm is 0.6-0.8, add 0.5 mol / L α-lactose to a final concentration of 0.03 mol / L, and culture at 37°C for another 6 h; (3) Collect the cells by centrifugation, add PBS (10 times the cell weight) to suspend the cells, disrupt them by ultrasonic wave, and collect the protein supernatant by centrifugation; (4) The supernatant was filtered through a 0.22 μm filter and purified using a nickel column. The purified protein was stored at -20°C until use.

Images