Preparation and application of porcine beta defensin compound preparation

Abstract

The invention relates to the field of animal breeding, and discloses a pig beta defensin compound preparation which comprises pig beta defensin PBD1, PBD2 and PBD114, and the weight ratio of the PBD1 to the PBD2 to the PBD114 is 1: 1: 1. The porcine beta defensin PBD1, the porcine beta defensin PBD2 and the porcine beta defensin PBD114 are recombinant proteins, and are respectively expressed in escherichia coli BL21 (DE3) PLysS competent cells by a pET-32a expression vector. Through the synergistic effect of the defensins PBD1, PBD2 and PBD114, the minimum inhibitory concentration (MIC) of the EPEC is reduced to 18.75 mu g / mL from 75 mu g / mL of single defensins, and is reduced by 75%; within 24 hours, the bacteriostasis rate of the compound preparation reaches 90%, and the inhibition effect on EPEC is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, and specifically to the preparation and application of a porcine β - defensin composite preparation. Background Art

[0002] In modern livestock and poultry breeding, the widespread use of traditional antibiotics has caused a series of problems, especially the emergence of drug resistance and drug residues. These problems not only threaten animal health but also pose potential risks to food safety and human health. Over a long period, bacteria have gradually developed stronger resistance to antibiotics, resulting in a reduced therapeutic effect of antibiotics, and even some bacteria have developed resistance to multiple antibiotics. In addition, the problem of antibiotic residues in meat, dairy products, and other animal products has also attracted great public attention to food safety.

[0003] To address these challenges, scientists have begun to search for new antibacterial substances to replace antibiotics. Among them, defensins, as a new type of antibacterial peptide, have shown great potential. Defensins are widely present in the immune systems of mammals and birds, have a strong antibacterial effect, and compared with traditional antibiotics, defensins have almost no drug resistance problems, do not cause drug residues, and are harmless to the human body.

[0004] Porcine β - defensin (PBD) has been proven to be highly expressed in the small intestine of pigs and has a significant antibacterial effect against a variety of pathogenic bacteria. In particular, porcine β - defensins PBD1, PBD2, and PBD114 have shown excellent antibacterial properties in many studies. However, existing research mainly focuses on the role of single defensins and lacks in - depth exploration of the synergistic effects between multiple porcine β - defensins. The existing research in the prior art mainly focuses on the antibacterial effects of single defensins, and their synergistic mechanism and optimal ratio have not been fully understood. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides the preparation and application of a porcine β - defensin composite preparation, which solves the problems of drug resistance and drug residues caused by traditional antibiotics in livestock breeding and provides a new antibacterial method by replacing antibiotics with a porcine β - defensin composite preparation.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions: A porcine β - defensin composite preparation, comprising porcine β - defensins PBD1, PBD2, and PBD114, wherein the weight ratio of PBD1, PBD2, and PBD114 is 1:1:1.

[0007] Preferably, the porcine β - defensins PBD1, PBD2, and PBD114 are recombinant proteins, which are respectively expressed by a pET - 32a expression vector in Escherichia coli BL21(DE3)PLysS competent cells.

[0008] A preparation method of a porcine β - defensin composite preparation, the method comprising the following steps: (a) Construct a recombinant expression vector through gene cloning technology, and clone the gene sequences of porcine β - defensins PBD1, PBD2, and PBD114 into the pET - 32a vector; (b) Transform the recombinant expression vector into Escherichia coli BL21(DE3)PLysS competent cells for induced expression; (c) Identify the expressed proteins using SDS - PAGE and Western Blot techniques; (d) Perform affinity purification using nickel column method and magnetic bead method to obtain purified PBD1, PBD2, and PBD114 proteins.

[0009] Preferably, the recombinant proteins PBD1, PBD2, and PBD114 are purified by affinity purification using a nickel column, and the concentrations of the purified proteins are 631 μg / mL, 636 μg / mL, and 360 μg / mL respectively.

[0010] Preferably, the gene sequences of PBD1, PBD2, and PBD114 are obtained from the GenBank database respectively and optimized according to the codon preference of Escherichia coli.

[0011] Preferably, the antibacterial effect of the composite preparation is tested by a micro - bacteriostatic test, and evaluated using pathogenic bacteria such as enteropathogenic Escherichia coli, ETEC, S. typhimurium, S. aureus, and S. sonnei as indicator bacteria.

[0012] Preferably, the minimum inhibitory concentration of the composite preparation is 18.75 μg / mL, which is used to inhibit the growth of enteropathogenic Escherichia coli.

[0013] Preferably, the expression temperature of the recombinant proteins PBD1, PBD2, and PBD114 is 37°C, the concentration of the inducer is 1 mM IPTG, and the expression time is 4 hours.

[0014] Preferably, the method further includes observing the changes in the cell ultrastructure of enteropathogenic Escherichia coli by electron microscopy to confirm the inhibitory effect of the composite preparation on enteropathogenic Escherichia coli.

[0015] An application of a porcine β - defensin composite preparation for preventing and treating piglet diarrhea caused by enteropathogenic Escherichia coli and having no risk of drug residues.

[0016] The present invention provides a preparation and application of a porcine β - defensin composite preparation. It has the following beneficial effects: 1. Through the synergistic effect of defensins PBD1, PBD2, and PBD114, the minimum inhibitory concentration (MIC) of EPEC was reduced from 75 μg / mL of a single defensin to 18.75 μg / mL, a 75% reduction. Within 24 hours, the bacteriostatic rate of the composite preparation reached 90%, significantly enhancing the inhibitory effect on EPEC and providing a more efficient alternative for antibacterial treatment.

[0017] 2. The present invention avoids the selective pressure generated during the use of traditional antibiotics. Through a multi-target membrane disruption mechanism, it effectively delays the development of drug resistance. The in vivo experimental results show that after using this composite preparation, the diarrhea rate of piglets decreased significantly, the intestinal bacterial load decreased by an order of magnitude, and there was no risk of drug residues, further ensuring food safety and animal health.

[0018] 3. As a green antibacterial alternative, the present invention can effectively replace the use of traditional antibiotics, reduce the dependence on antibiotics in the aquaculture industry, and reduce the risk of environmental pollution. This technology conforms to the development trend of green agriculture and contributes to promoting sustainable aquaculture and reducing environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of the steps of the present invention; Figure 2 is a schematic diagram of the experimental principle of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Please refer to the attached Figure 1 - attached Figure 2 , the embodiment of the present invention provides a preparation method of a porcine β-defensin composite preparation, including the following steps: (a) Construct a recombinant expression vector through gene cloning technology, and clone the gene sequences of porcine β-defensins PBD1, PBD2, and PBD114 into the pET-32a vector; (b) Transform the recombinant expression vector into Escherichia coli BL21(DE3)PLysS competent cells for induction expression; (c) Identify the expressed protein using SDS-PAGE and Western Blot techniques; (d)The nickel column method and magnetic bead method were used for affinity purification to obtain purified PBD1, PBD2, and PBD114 proteins.

[0022] Specifically, the gene sequences of PBD1, PBD2, and PBD114 were obtained from the GenBank database. According to the codon preference of Escherichia coli, the codons were optimized and then cloned into the pET-32a expression vector to construct recombinant expression vectors.

[0023] They were transformed into BL21(DE3)PLysS competent cells for protein expression. The expressed proteins were identified by SDS-PAGE and Western Blot techniques, and the target proteins were purified by the magnetic bead method. The results showed that the recombinant expression vectors pET32a-PBD1, pET32a-PBD2, and pET32a-PBD114 were successfully constructed, and recombinant proteins PBD1, PBD2, and PBD114 with sizes of approximately 22.2, 22.1, and 26.0 kDa respectively were obtained after induction.

[0024] The recombinant proteins were affinity purified using nickel columns, and the concentrations of the purified recombinant proteins were measured to be 631, 636, and 360 μg / mL respectively by a BCA protein concentration assay kit.

[0025] To verify the in vitro antibacterial activities of the three recombinant proteins, five pathogenic bacteria, namely Salmonella typhimurium LT2 strain, Shigella sonnei strain ATCC29930, Staphylococcus aureus strain NBRC100910, enteropathogenic Escherichia coli EPEC CICC24189, and enterotoxigenic Escherichia coli ETEC BNCC125783, were used as indicator strains. Through the MIC microbacteriostatic test, it was shown that the MIC of PBD1 against EPEC was 75 μg / mL, the MIC against ETEC and S. typhimurium were 150 μg / mL and 150 μg / mL respectively, and there was no antibacterial effect on S. aureus and S. sonnei or the minimum inhibitory concentration > 150 μg / mL; The MIC of PBD2 against both EPEC and ETEC was 75 μg / mL, the MIC against S. typhimurium was 150 μg / mL, there was no antibacterial effect on S. aureus or the minimum inhibitory concentration > 150 μg / mL, and the MIC against S. sonnei was 37.5 μg / mL; The MIC of PBD114 against EPEC, ETEC, and S. typhimurium was 75 μg / mL, and the MIC against S. aureus and S. sonnei was 150 μg / mL.

[0026] Three porcine β - defensins were compounded in multiple ratios and grouped. According to PBD1:PBD2:PBD114, groups of 0:1:1, 1:0:1, 1:1:0, 1:1:1, 1:1:2, 1:2:1, and 2:1:1 were prepared respectively. The MIC micro - bacteriostatic experiment was carried out on each group to detect the minimum inhibitory concentration of the 7 groups of ratios against 5 kinds of pathogenic bacteria, and then the FIC index was calculated. The results showed that the three defensins had a synergistic effect on EPEC, and the MIC of the 1:1:1 group against EPEC was the smallest, and the synergistic effect was the most obvious; the three defensins had an antagonistic effect on Shigella sonnei strain ATCC29930 and Staphylococcus aureus strain NBRC100910; the three defensins had no synergistic or inhibitory interaction on ETEC and Salmonella typhimurium LT2 strain.

[0027] According to the results, the best ratio of the porcine β - defensin compound preparation was selected as 1:1:1. To verify its potential as a new antibacterial drug, its bacteriostatic effect was further evaluated. The minimum inhibitory concentration of the compound preparation against EPEC was verified to be 18.75 μg / mL by the dilution spot - plate method; the Kill - Time killing curve was drawn to prove that the compound preparation had an inhibitory effect on EPEC within 24 h but no bactericidal effect; through the electron microscope observation of the bacterial ultrastructure, it was found that the morphological changes of EPEC occurred significantly after the action of the compound preparation, showing shrinkage, swelling or deformation, rough surface, and local depressions, holes or damaged membrane structures could be seen.

[0028] To verify the in - vivo bacteriostatic activity of the compound preparation, an infection model of EPEC in Bama miniature pigs was established, and the optimal challenge concentration of piglets against EPEC was determined to be 1×108 CFU / mL. After challenging and administering the compound preparation to the piglets, the status of the piglets was observed, fecal scoring was carried out, blood routine examination was performed, autopsy was observed for lesions, immune organ index was calculated, small intestine sections were prepared, intestinal bacterial load was calculated, and the expression levels of intestinal cytokine - related and signal transduction - related genes were measured. The results showed that the compound preparation alleviated the adverse effects of EPEC on piglets in all indicators.

[0029] Through the pre - experiment, it was proved that the three - PBD compound preparation had a significant inhibitory effect on EPEC and could effectively treat the piglet infection caused by EPEC. When exploring the changes in the expression levels of PBD1, PBD2, and PBD114 genes in the small intestine of Bama miniature pigs before and after EPEC infection, it was found that after piglets were infected with EPEC, the expression levels of these three PBD genes in the jejunum increased significantly. This phenomenon indicates that the three PBDs are very likely to be the key pathways for the body to resist EPEC infection. This also enhances the theoretical basis for the three - PBD compound preparation to be used as a potential new drug from another perspective.

[0030] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A porcine β - defensin composite preparation, characterized in that, The porcine β-defensins PBD1, PBD2 and PBD114, where the weight ratio of PBD1, PBD2, and PBD114 is 1:1:

1.

2. The porcine β-defensin composite preparation according to claim 1, wherein The porcine β-defensins PBD1, PBD2 and PBD114 are recombinant proteins, which are respectively expressed by the pET-32a expression vector in Escherichia coli BL21(DE3)PLysS competent cells.

3. A preparation method of a porcine β - defensin composite preparation, according to the porcine β - defensin composite preparation described in any one of claims 1 - 2, characterized in that, The method includes the following steps: (a) Construct a recombinant expression vector through gene cloning technology, and clone the gene sequences of porcine β-defensins PBD1, PBD2 and PBD114 into the pET-32a vector; (b) Transform the recombinant expression vector into Escherichia coli BL21(DE3)PLysS competent cells for induced expression; (c) Identify the expressed proteins using SDS-PAGE and Western Blot techniques; (d) Perform affinity purification using nickel column method and magnetic bead method to obtain purified PBD1, PBD2 and PBD114 proteins.

4. The preparation method of a porcine β - defensin composite preparation according to claim 3, characterized in that, The purification of the recombinant proteins PBD1, PBD2, PBD114 is carried out by affinity purification using nickel column, and the concentrations of the purified proteins are 631 μg / mL, 636 μg / mL, and 360 μg / mL respectively.

5. A preparation method of a porcine β - defensin composite preparation according to claim 3, characterized in that, The gene sequences of PBD1, PBD2, PBD114 are respectively obtained according to the GenBank database and optimized according to the codon preference of Escherichia coli.

6. The preparation method of a porcine β - defensin composite preparation according to claim 3, characterized in that, The antibacterial effect of the composite preparation is tested by micro-bacteriostatic test, and evaluated using pathogenic bacteria such as enteropathogenic Escherichia coli, ETEC, S. typhimurium, S. aureus and S. sonnei as indicator bacteria.

7. A preparation method of a porcine β - defensin composite preparation according to claim 3, characterized in that, The minimum inhibitory concentration of the composite preparation is 18.75 μg / mL, which is used to inhibit the growth of enteropathogenic Escherichia coli.

8. A preparation method of a porcine β - defensin composite preparation according to claim 3, characterized in that, The expression temperature of the recombinant proteins PBD1, PBD2 and PBD114 is 37°C, the inducer concentration is 1 mM IPTG, and the expression time is 4 hours.

9. The preparation method of a porcine β - defensin composite preparation according to claim 3, characterized in that, The method further includes observing the changes in the cell ultrastructure of enteropathogenic Escherichia coli by electron microscopy to confirm the inhibitory effect of the composite preparation on enteropathogenic Escherichia coli.

10. Use of a porcine β-defensin composite preparation, the porcine β-defensin composite preparation according to any one of claims 1-2, characterized in that, It is used for preventing and treating piglet diarrhea caused by enteropathogenic Escherichia coli, and there is no risk of drug residue.

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