Antibacterial recombinant protein as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial recombinant protein as well as a preparation method and application thereof. The antibacterial recombinant protein provided by the invention has excellent antibacterial activity, can realize efficient production, and can be used for industrial large-scale production and preparation. The antibacterial recombinant protein disclosed by the invention can be applied to the fields of feeds, medicines, preservatives, functional foods, biopesticides, skin care products or antibacterial coatings and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial proteins, and in particular relates to an antibacterial recombinant protein and a preparation method and application thereof. Background Art

[0002] Since the ban on antibiotics in the feed industry, the research and development and application of antimicrobial peptides as alternatives to antibiotics have become a hot topic. Porcine β-defensin 1 is an antimicrobial peptide found in pigs. Its structure contains three disulfide bonds, which form a rigid structure that resists degradation by proteases. Porcine β-defensin 1 has a strong inhibitory effect on a variety of bacteria, including multidrug-resistant strains, and has some antiviral activity. Porcine β-defensin 1 can promote piglet growth, improve sow body condition, and regulate pig immune responses. Porcine β-defensin 1 can also participate in influencing multiple cell signaling pathways, indirectly affecting processes such as pig inflammatory responses.

[0003] However, despite their numerous advantages, antimicrobial peptides still face significant challenges in their application. On the one hand, the high cost of expressing antimicrobial peptides limits their large-scale production; on the other hand, their antibacterial efficacy and safety need to be further improved. Therefore, research and development focused on modifying and expanding the functionality of antimicrobial peptides to optimize their function is crucial. Summary of the Invention

[0004] To solve at least some of the technical problems in the above-mentioned prior art, the present invention provides an antibacterial recombinant protein and its preparation method and application. Specifically, the present invention includes the following contents.

[0005] The first aspect of the present invention provides an antibacterial recombinant protein, comprising:

[0006] (1) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO. 1 and has the same function; or

[0007] (2) An amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1 and having the same function.

[0008] In certain embodiments, the antibacterial recombinant protein according to the present invention has an amino acid sequence shown in SEQ ID NO.1.

[0009] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibacterial recombinant protein according to the present invention.

[0010] The third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule according to the present invention.

[0011] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or vector molecule according to the present invention.

[0012] In a fifth aspect, the present invention provides a method for preparing the antibacterial recombinant protein according to the present invention, wherein the antibacterial recombinant protein is prepared by artificial synthesis or genetic engineering.

[0013] In a sixth aspect, the present invention provides an antibacterial composition comprising the antibacterial recombinant protein according to the present invention and excipients.

[0014] In a seventh aspect, the present invention provides use of the antibacterial recombinant protein according to the present invention in the preparation of antibacterial products.

[0015] In certain embodiments, according to the use of the present invention, the antibacterial product includes feed, medicine, preservative, functional food, biopesticide, skin care product or antibacterial coating.

[0016] In an eighth aspect, the present invention provides a method for inhibiting bacteria, comprising the step of using the antibacterial recombinant protein according to the present invention.

[0017] In certain embodiments, the method for inhibiting bacteria according to the present invention is an in vitro method.

[0018] In genetically engineered expression systems, exogenous proteins can be reduced in activity, unable to be expressed or secreted, due to factors such as the properties of the exogenous gene, protease degradation, protein misfolding, and protein modifications. To achieve normal secretory expression of antimicrobial peptides in genetically engineered expression systems, the present invention has constructed an antimicrobial recombinant protein. This antimicrobial recombinant protein exhibits excellent antibacterial activity, enables efficient production, and is suitable for industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the PCR verification of porcine β-defensin 1 (pBD1) and LP-pBD1 target gene fragments, wherein the marker lane is a DNA marker, the first lane is the pBD1 gene, and the second lane is the LP-pBD1 gene.

[0020] Figure 2 The figure shows the SDS-PAGE electrophoresis results of the supernatant of the Pichia pastoris protein induced expression, wherein lane M is a protein marker, and lanes 1, 2, and 3 are the supernatants of three Pichia pastoris transformed with the pBD1 gene sequence after 96 hours of induction.

[0021] Figure 3The figure shows the SDS-PAGE electrophoresis results of the supernatant of the Pichia pastoris protein induced expression, wherein lane M is a protein marker, and lanes 1, 2, and 3 are the supernatants of three Pichia pastoris transformed with the LP-pBD1 gene sequence after 96 hours of induction.

[0022] Figure 4 The map of the pPICZαA plasmid containing the recombinant antimicrobial peptide protein gene PIGF-LP-pBD1 is shown.

[0023] Figure 5 The figure shows the results of enzyme digestion verification of the pPICZαA plasmid containing the recombinant antimicrobial peptide protein gene Gox-LP-pBD1, wherein the marker lane is a DNA marker, and the last two lanes are double enzyme digestion verification of two groups of pPICZαA plasmids containing the recombinant antimicrobial peptide protein gene using NotI and EcoRI.

[0024] Figure 6 The figure shows the results of enzyme digestion verification of the pPICZαA plasmid containing the recombinant antimicrobial peptide protein gene survivin-LP-pBD1, wherein the marker lane is a DNA marker, and the last two lanes are double enzyme digestion verification of two groups of pPICZαA plasmids containing the recombinant antimicrobial peptide protein gene using NotI and EcoRI.

[0025] Figure 7 The figure shows the results of enzyme digestion verification of the pPICZαA plasmid containing the recombinant antimicrobial peptide protein gene PIGF-LP-pBD1, wherein the marker lane is a DNA marker, and the last two lanes are double enzyme digestion verification of two groups of pPICZαA plasmids containing the recombinant antimicrobial peptide protein gene using NotI and EcoRI.

[0026] Figure 8 The figure shows the screening of transformed Pichia pastoris cells using YPD medium containing high concentration of Zeocin antibiotic (Zeocin concentration is 2000 μg / mL), wherein the monoclonal colony grown on the left is the Pichia pastoris strain containing multiple copies of the target gene.

[0027] Figure 9 The figure shows the PCR verification of Pichia pastoris strain protein before induction, wherein the marker lane is a DNA marker, and lanes 1 and 2 are two strains integrated with the recombinant antimicrobial peptide protein PIGF-LP-pBD1 gene.

[0028] Figure 10Shown are SDS-PAGE electrophoresis results of the supernatant from Pichia pastoris transformed with the recombinant antimicrobial peptide protein gene Gox-LP-pBD1 after protein expression induction. Lane Marker represents a protein marker, while lanes 1, 2, and 3 represent the supernatants from three Pichia pastoris strains transformed with the Gox-LP-pBD1 gene sequence after 96 hours of induction.

[0029] Figure 11 Shown are SDS-PAGE electrophoresis results of the supernatant from Pichia pastoris transformed with the recombinant antimicrobial peptide gene survivin-LP-pBD1 after protein expression. Lane Marker represents a protein marker, while lanes 1, 2, and 3 represent the supernatants from three Pichia pastoris strains transformed with the survivin-LP-pBD1 gene sequence after 96 hours of induction.

[0030] Figure 12 Shown are the results of SDS-PAGE electrophoresis of the supernatant from Pichia pastoris transformed with the recombinant antimicrobial peptide gene PIGF-LP-pBD1 after protein expression. Lane Marker represents a protein marker, while lanes 1, 2, and 3 represent the supernatants from three Pichia pastoris strains transformed with the PIGF-LP-pBD1 gene sequence after 96 hours of induction.

[0031] Figure 13 Figure 2 shows the antibacterial effects of lyophilized powders of supernatant from Pichia pastoris transfected with the recombinant antimicrobial peptide proteins Gox-LP-pBD1 and survivin-LP-pBD1 after 96 hours of induction. The wells labeled "1, 2, and 3" represent the single-, double-, and triple-concentration groups, respectively, obtained by dissolving the lyophilized powders in pure water at their original concentrations. The well labeled "A" represents the positive control group containing 100 μg / mL ampicillin. The left side shows the antibacterial effect of the recombinant protein Gox-LP-pBD1, and the right side shows the antibacterial effect of the recombinant protein survivin-LP-pBD1.

[0032] Figure 14 The antibacterial results of the lyophilized powder of the bacterial supernatant after 96 hours of induction of Pichia pastoris transferred with the recombinant antimicrobial peptide protein gene PIGF-LP-pBD1 are shown. Among them, the sample wells marked "1×, 2×, 3×" are the one-fold, two-fold and three-fold concentrated groups prepared by dissolving the lyophilized powder in pure water at the original concentration, respectively. The sample well marked "A" is the positive control group containing 100 μg / mL ampicillin, and the sample well marked "Ci" is the supernatant of the fermentation broth with an empty vector.

[0033] Figure 15The antibacterial results of the lyophilized powder of the bacterial supernatant after 96 hours of induction of Pichia pastoris transferred with the recombinant antimicrobial peptide protein gene LP-pBD1 are shown. Among them, the sample wells marked "1×, 2×, 3×" are the one-fold, two-fold, and three-fold concentrated groups prepared by dissolving the lyophilized powder in pure water at the original concentration, respectively. The sample well marked "A" is the positive control group containing 100 μg / mL ampicillin. The sample well marked "Ci" is the supernatant of the fermentation broth with an empty vector.

[0034] Figure 16 The antibacterial effects of the recombinant antimicrobial proteins LP-pBD1 and PIGF-LP-pBD1 are shown. The comparison samples are reconstituted solutions of the lyophilized powder of the fermentation supernatant of the three-fold concentrated recombinant antimicrobial protein LP-pBD1 and PIGF-LP-pBD1. From left to right in the figure are Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Salmonella (S. enterica). DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0038] Antibacterial recombinant protein

[0039] One aspect of the present invention provides an antibacterial recombinant protein, comprising:

[0040] (1) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO. 1 and has the same function; or

[0041] (2) An amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1 and having the same function.

[0042] In a preferred embodiment, the antibacterial recombinant protein has the amino acid sequence shown in SEQ ID NO.1.

[0043] In the present invention, "antibacterial" refers to inhibiting bacterial growth or activity, inhibiting bacterial proliferation or directly killing bacteria. Wherein, the bacteria include Gram-positive bacteria and Gram-negative bacteria. Wherein, examples of Gram-positive bacteria include but are not limited to Staphylococcus (such as but not limited to Staphylococcus aureus, Staphylococcus epidermidis, etc.), Streptococcus (such as but not limited to Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, etc.), Enterococcus (such as but not limited to Enterococcus faecalis, Enterococcus faecium, etc.), Bacillus (such as but not limited to Bacillus anthracis, Bacillus subtilis, etc.), Listeria (such as but not limited to Listeria monocytogenes), Corynebacterium (such as but not limited to Corynebacterium diphtheriae), Actinomyces (such as but not limited to Actinomyces ileri), Mycobacteria (such as but not limited to Mycobacterium tuberculosis, Mycobacterium leprae, non-tuberculous mycobacteria, etc.), Erysipelothrix (such as but not limited to Erysipelothrix rhizogenes), etc. Examples of Gram-negative bacteria include, but are not limited to, Salmonella, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas fluorescens, Proteus, Shigella, Yersinia, Neisseria, Haemophilus, Legionella, Brucella, Bordetella, and the like.

[0044] In the present invention, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. To determine sequence identity, a sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithm required for achieving optimal alignment in the full-length sequences being compared.

[0045] In the present invention, antibacterial recombinant protein sequences obtained by modification, substitution, deletion, or addition of one or more amino acids also fall within the scope of protection of the present invention. The term "modification" refers to any chemical modification of an amino acid sequence. The term "substitution" refers to the replacement of one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results in the addition of one or more amino acids compared to the naturally occurring molecule.

[0046] In the present invention, the method for detecting the antibacterial effect is not particularly limited and can be performed using methods known in the art, such as the Oxford cup method, filter paper method, inhibition ring method, minimum inhibitory concentration determination, time-kill curve method, etc.

[0047] In genetic engineering expression systems, the activity of the antimicrobial peptide porcine β-defensin 1 is reduced, it cannot be expressed or secreted due to the characteristics of exogenous genes, protease degradation, protein folding errors, protein mismodification, etc. In order to achieve the normal secretory expression of β-defensin 1 in a genetic engineering expression system, the present invention constructs a short antimicrobial peptide with a linear structure, namely a linker peptide (LP). Constructing a linker peptide to the N-terminus of β-defensin 1 can ensure that the protein structure of β-defensin 1 is correctly folded while enhancing the antibacterial activity of β-defensin 1. However, after being connected with the linker peptide, the antimicrobial activity of the antimicrobial peptide porcine β-defensin 1 is still weak, and the expression level is low. Therefore, the present invention attempts to connect different proteins to antimicrobial peptides through a large number of screening experiments to improve the activity of the antimicrobial peptide protein. The present invention studies the simultaneous connection of glucose oxidase (Gox), survivin, placental growth factor (PIGF) and a connecting peptide to the front end of β-defensin 1. The results show that glucose oxidase and survivin have no effect on improving the activity of antimicrobial peptides, while connecting placental growth factor to the front end of the recombinant antimicrobial peptide can greatly enhance the antimicrobial activity of the antimicrobial peptide porcine β-defensin 1 and increase the expression level, thereby making it suitable for industrial scale-up production and preparation.

[0048] In a preferred embodiment, the connecting peptide has the sequence shown in SEQ ID NO.3.

[0049] Nucleic acid molecules

[0050] In one aspect of the present invention, a nucleic acid molecule is provided, comprising a nucleotide sequence encoding an antibacterial recombinant protein according to the present invention. As used herein, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length, comprising deoxyribonucleotides, ribonucleotides, and / or analogs thereof, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Thus, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, and the like.

[0051] The nucleic acid molecules of the present invention include coding sequences that have been codon-optimized. A codon is a group of three adjacent nucleotides in a messenger RNA molecule that represents a specific amino acid during protein synthesis. "Codon optimization" is intended to include altering the codon composition of a recombinant nucleic acid without changing the amino acid sequence.

[0052] In a preferred embodiment, the nucleotide sequence has the sequence shown in SEQ ID NO.2.

[0053] Once the coding sequence of the antibacterial recombinant protein of the present invention is isolated, the antibacterial recombinant protein can be obtained in large quantities using recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then isolate it from the propagated host cells using conventional methods.

[0054] Carrier molecules

[0055] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0056] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector includes a target gene, a promoter, a terminator encoding the antibacterial recombinant protein of the present invention, or optionally further includes a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0057] In a preferred embodiment, the recombinant vector has the sequence shown as SEQ ID NO.4.

[0058] host cells

[0059] In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule of the present invention.

[0060] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. Host cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0061] Preparation method

[0062] One aspect of the present invention provides a method for preparing the antibacterial recombinant protein of the present invention. The preparation method is not particularly limited and includes preparation by artificial synthesis or genetic engineering.

[0063] In certain embodiments, the antibacterial recombinant protein of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing antibacterial recombinant proteins are known in the art, for example, the antibacterial recombinant protein of the present invention can be obtained by direct amino acid synthesis.

[0064] In certain embodiments, the antibacterial recombinant protein of the present invention is obtained by genetic engineering expression. Genetic engineering expression systems used for genetic engineering expression include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include, but are not limited to, Escherichia coli expression systems. Eukaryotic cell expression systems include, but are not limited to, zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0065] In a preferred embodiment, the antibacterial recombinant protein of the present invention can be prepared by the following steps:

[0066] (1) constructing a recombinant vector containing an antibacterial recombinant protein expressing the amino acid sequence shown in SEQ ID NO.1;

[0067] (2) transforming the vector into host cells and culturing the cells under conditions suitable for the expression of the antibacterial recombinant protein;

[0068] (3) Collect and purify the antibacterial recombinant protein.

[0069] Antibacterial composition

[0070] In one aspect, the present invention provides an antimicrobial composition comprising the antimicrobial recombinant protein according to the present invention and an excipient. The excipient can be any suitable carrier or excipient used in the production or preparation of feed, medicine, preservative, functional food, biopesticide, skin care product, or antimicrobial coating, and examples thereof include, but are not limited to, at least one of a diluent, a filler, an absorbent, a wetting agent, an adhesive, a preservative, and an antioxidant. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, calcium hydrogen phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; adhesives include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0071] application

[0072] One aspect of the present invention provides use of the antibacterial recombinant protein according to the present invention in the preparation of antibacterial products.

[0073] In the present invention, examples of the antibacterial products include but are not limited to feed, medicine, preservatives, functional foods, biological pesticides, skin care products, antibacterial coatings, etc.

[0074] Methods of inhibiting bacteria

[0075] In one aspect, the present invention provides a method for inhibiting bacteria, comprising the step of using the antimicrobial recombinant protein described herein. Applications thereof include, but are not limited to, equipment and environmental disinfection, preparation of antimicrobial materials, food processing, agricultural planting and breeding, and experimental research.

[0076] In a preferred embodiment, the method is an in vitro method.

[0077] In the present invention, "inhibit bacteria", "bacteriostatic" and "antibacterial" are used interchangeably and refer to inhibiting bacterial growth or activity, inhibiting bacterial proliferation or directly killing bacteria.

[0078] Example

[0079] This example shows the preparation process and antibacterial effect of the antibacterial recombinant protein.

[0080] 1. Expression and identification of porcine β-defensin pBD1

[0081] 1. Materials and Methods

[0082] 1.1 Materials and Instruments

[0083] The plasmids, Pichia pastoris X33 strain, and Escherichia coli, Staphylococcus aureus, and Salmonella strains used in this example were all obtained from Dong Na's laboratory at the College of Animal Science and Technology, China Agricultural University.

[0084] Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, methanol, and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd. Carrier DNA, X33 competent cells, lithium acetate, and Zeocin antibiotics were purchased from Beijing Coolbo Technology Co., Ltd. Biotin, YNB, sorbitol, and agar were purchased from Beijing Solebeau Technology Co., Ltd. PCR Mix and protein markers were purchased from Yisheng Biotechnology Co., Ltd. Primers were synthesized by Beijing Ruibo Xingke Co., Ltd. Yeast extract and peptone were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Enzymes NotI and EcoRI were purchased from New England Biolabs.

[0085] ZQZY-88CES, ZQZYB8ES, and ZQZYC8ES constant-temperature shaking incubators were purchased from Shanghai Zhichu Instrument Co., Ltd.; DHP-9052B constant-temperature incubator was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.; CWE811 PCR instrument was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; PowerPac300 electrophoresis instrument was purchased from Bio-Rad; TANON-1600 gel imaging system was purchased from Shanghai Tianneng Technology Co., Ltd.; 5424 desktop centrifuge was purchased from Eppendorf; NanoDrop Lite spectrophotometer was purchased from ThermoFisher; MLS-3781L-PC high-pressure steam autoclave was purchased from Panasonic; P2, P20, P200, and P1000 pipettes were purchased from Gilson; and DL-CJ-1NDII clean bench was purchased from Beijing Donglianhaer Instrument Manufacturing Co., Ltd.

[0086] 1.2 Experimental methods

[0087] 1.2.1 Construction and identification of expression vector

[0088] The porcine β-defensin 1 gene was codon-optimized to adapt to the expression preference of Pichia pastoris. The gene fragment was synthesized by Beijing Ruibo Xingke Co., Ltd. and spliced ​​into the pPICZαA plasmid vector to construct the recombinant expression plasmid pPICZαA-pBD1.

[0089] The company's synthesized recombinant plasmid pPICZαA-pBD1 was transformed into Escherichia coli DH5α competent cells. The transformed E. coli competent cells were plated on 2YT solid medium containing Zeocin antibiotics and incubated for 12 hours. A single E. coli colony of appropriate size was selected and inoculated into 5 mL of 2YT medium (containing 100 μg / mL Zeocin). The culture was incubated at 37°C and 220 rpm for 12 hours for plasmid extraction. The recombinant plasmid was double-digested with EcoRI and NotI (see Table 1 for the reaction system). The enzyme digestion was performed at 37°C for 2 hours, and 10 μL was subjected to 1% agarose gel electrophoresis. The recombinant plasmid was sequenced, and the sequence obtained was aligned with the optimized sequence using Snapgene software. The plasmid with the correct sequencing and enzyme digestion results was selected for transformation.

[0090] 1.2.2 Expression and identification of recombinant antimicrobial proteins in Pichia pastoris

[0091] The recombinant plasmid was digested with enzymes according to the system shown in Table 1. The reaction system was reacted at 37°C for 2 h, and the fragments were recovered. The linearized plasmid was dissolved in sterile enzyme-free water for later use.

[0092] Table 1 Recombinant plasmid linearization system

[0093]

[0094] Mix 30 μL of linearized plasmid with 10 μL of pre-denatured carrier DNA and add to 200 μL of unthawed competent cells. Place the mixture in a 30°C water bath and invert every 15 seconds until the competent cells are just thawed, then remove. Add 1.4 mL of B2 solution to the mixture and invert to mix thoroughly. Incubate in a 30°C water bath for 60 minutes, inverting every 20 minutes to mix thoroughly. Centrifuge at 3000 rpm for 3 minutes, discard the supernatant, and resuspend the cells in 1 mL of B3 solution. Centrifuge at 3000 rpm for 3 minutes, discard the supernatant, and resuspend the cells in 1 μL of B3 solution. Spread 100 μL of the bacterial solution onto Zeocin-resistant YPD medium and incubate at 30°C for 3 days until yeast colonies grow in the YPD medium.

[0095] From the YPD culture containing successfully transformed yeast, single colonies were selected and transferred to a 100 mL Erlenmeyer flask containing 10 mL of YPD liquid medium supplemented with 100 μg / mL Zeocin antibiotic. Culture was performed at 28°C, 220 rpm, for 24 hours. Next, 100 μL of the culture was transferred to a 1.5 mL centrifuge tube and frozen in liquid nitrogen for 5 minutes. The tube was then transferred to a 95°C metal bath and heated for 5 minutes. After heating, the tube was transferred to liquid nitrogen and frozen again. This process was repeated three times. PCR analysis was then performed using the frozen-thawed culture. The PCR reaction system is shown in Table 2. 10 μL of the PCR product was subjected to 1% agarose gel electrophoresis.

[0096] Table 2 PCR identification system

[0097]

[0098] Table 3 Primer sequences used in the experiment

[0099]

[0100]

[0101] A single identified colony was picked and inoculated into 10 mL of YPD liquid medium and shaken at 220 rpm at 28°C for 24 hours. Then, 100 μL of the culture was inoculated into 50 mL of BMGY medium and cultured until the OD600 reached 2.0. The culture was then placed in a 50 mL centrifuge tube and centrifuged at 4000 rpm at 4°C for 15 minutes. The supernatant was discarded, and the pellet was resuspended in BMMY medium and transferred to 50 mL of BMMY medium for induction. Methanol was added every 12 hours for four days. The initial methanol concentration in the medium was 0.5%, and the amount of methanol added was increased gradually, to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%. The supernatant was collected every 24 hours, and 5× loading buffer was added to the sample. The sample was heated in a 95°C metal bath for 40 minutes. The sample was then subjected to 12% SDS-PAGE electrophoresis to determine the protein content in the supernatant.

[0102] 2. Results Analysis

[0103] 2.1PCR identification of target gene fragments

[0104] like Figure 1 As shown, the pPICZαA plasmid carrying the target gene pBD1 was successfully obtained through molecular cloning. The first lane in the figure is the PCR verification result of the plasmid. After electrophoresis, the size of the gene band is about 126bp, which is consistent with the size of the target gene.

[0105] 2.2 Detection of Pichia pastoris expression products

[0106] After three days of fermentation, the supernatant of the bacterial solution was subjected to SDS-PAGE electrophoresis analysis. Figure 2 As shown in the figure, after staining with Coomassie Brilliant Blue and destaining with destaining buffer, no obvious band was observed at the expected protein size. The antimicrobial peptide pBD1 was not successfully expressed and secreted.

[0107] 2. Expression and identification of recombinant antimicrobial peptide LP-pBD1

[0108] 1. Materials and Methods

[0109] 1.1 Materials, reagents, and instruments are the same as above

[0110] 1.2 Experimental methods

[0111] Based on the amino acid sequence of porcine β-defensin pBD1 from Experiment 1 (Expression and Identification of Porcine β-Defensin pBD1), a linker peptide LP was added to the N-terminus of pBD1 to create the recombinant antimicrobial peptide LP-pBD1. The LP-pBD1 gene sequence was codon-optimized using a codon optimization website to adapt to the expression preference of Pichia pastoris. This gene fragment was synthesized by Beijing Ruibo Xingke Co., Ltd. and spliced ​​into the pPICZαA plasmid vector to construct the recombinant expression plasmid pPICZαA-LP-pBD1.

[0112] Table 4LP amino acid sequence

[0113]

[0114] The acquisition and identification of the recombinant plasmid, and the expression and identification of the recombinant antimicrobial peptide protein in Pichia pastoris were the same as those in Experiment 1.

[0115] 2. Results Analysis

[0116] 2.1PCR identification of target gene fragments

[0117] like Figure 1 As shown, the pPICZαA plasmid carrying the target gene LP-pBD1 was successfully obtained through molecular cloning. The second lane in the figure is the PCR verification result of the plasmid, and the result is about 228bp, which is consistent with the size of the target gene.

[0118] 2.2 Detection of Pichia pastoris expression products

[0119] After three days of fermentation, the supernatant of the bacterial solution was analyzed by SDS-PAGE electrophoresis. Figure 3As shown, after staining with Coomassie Brilliant Blue and decolorizing with a decolorizing solution, a distinct band at approximately 8.1 kDa was observed, indicating that the recombinant antimicrobial peptide LP-pBD1 was successfully expressed and secreted in Pichia pastoris. Three distinct protein bands were observed in the supernatant. It is speculated that the three bands are due to the properties of disulfide bonds, which are disrupted by reductases in the endoplasmic reticulum of Pichia pastoris, leading to misfolding. However, most antimicrobial peptides form stable disulfide bonds, folding correctly into stable structures with antibacterial activity. Recombinant antimicrobial peptide LP-pBD1 demonstrated a small amount of antibacterial activity in subsequent validation.

[0120] 3. Expression and identification of recombinant antimicrobial peptides Gox-LP-pBD1, survivin-LP-pBD1, and PIGF-LP-pBD1

[0121] 1. Materials and Methods

[0122] 1.1 Materials are the same as those in Experiment 1

[0123] 1.2 Experimental Methods

[0124] 1.2.1 Construction and identification of expression vector

[0125] Based on the amino acid sequence of the recombinant antimicrobial peptide LP-pBD1 expressed and identified in Experiment 2, three different proteins—glucose oxidase Gox, survivin, and growth factor PlGF—were added to the N-terminus of the recombinant antimicrobial peptide to create the recombinant antimicrobial peptides Gox-LP-pBD1, survivin-LP-pBD1, and PlGF-LP-pBD1. The codon optimization website was used to codon-optimize the Gox-LP-pBD1, survivin-LP-pBD1, and PlGF-LP-pBD1 gene sequences to adapt them to the expression preferences of Pichia pastoris. The gene fragments were synthesized by Beijing Ruibo Xingke Co., Ltd. and spliced ​​into the pPICZαA plasmid vector to construct the recombinant expression plasmids pPICZαA-Gox-LP-pBD1, pPICZαA-survivin-LP-pBD1, and pPICZαA-PlGF-LP-pBD1. Figure 4 Shown is the map of the pPICZαA plasmid containing the recombinant antimicrobial peptide protein gene PIGF-LP-pBD1, which is the final practical application plasmid.

[0126] Table 5 Amino acid sequences of different recombinant proteins

[0127]

[0128]

[0129] The acquisition and identification of the recombinant plasmid, and the expression and identification of the recombinant antimicrobial peptide protein in Pichia pastoris were the same as those in Experiment 1.

[0130] 1.2.2 Screening of High-Copy Pichia Pastoris and Induced Expression in Shake Flasks

[0131] After identification, single yeast colonies were inoculated into a medium containing a high concentration of Zeocin (2000 μg / mL). After four days of culture, well-growing colonies were selected and streaked. A single colony was picked from a YPD plate containing high-copy yeast and inoculated into 10 mL of YPD liquid medium. The culture was shaken at 220 rpm at 28°C for 24 hours. 100 μL of the culture was then inoculated into 50 mL of BMGY medium and cultured until the OD600 reached 2.0. The culture was then transferred to a 50 mL centrifuge tube and centrifuged at 4000 rpm at 4°C for 15 minutes. The supernatant was discarded, and the pellet was resuspended in BMMY medium and transferred to 50 mL of BMMY medium for induction. Methanol was added every 12 hours for four days. The initial methanol concentration in the medium was 0.5%, and methanol was added in increasing amounts each time: 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%. The supernatant was collected every 24 hours, 5× loading buffer was added to the sample, and the sample was heated in a 95° C. metal bath for 40 minutes. The sample was then subjected to 12% SDS-PAGE electrophoresis to detect the protein content in the supernatant.

[0132] The fermented bacterial liquid was divided into centrifuge tubes, centrifuged at 4°C, 4000 rpm for 1 hour, the supernatant was poured out, and the liquid was placed in a freeze dryer for freeze drying for three days. After three days, the freeze-dried powder was recovered and weighed.

[0133] 2. Experimental Results

[0134] 2.1 Verification of recombinant expression plasmid

[0135] The constructed recombinant expression plasmid was double-enzyme digested and verified. The results showed that the target gene was successfully constructed into the pPICZαA plasmid. The enzyme digestion results of pPICZαA-Gox-LP-pBD1, pPICZαA-survivin-LP-pBD1 and pPICZαA-PIGF-LP-pBD1 were as follows: Figure 5-Figure 7 As shown, the target gene fragments are 1998 bp, 711 bp, and 774 bp, respectively. The sequencing results also show that the plasmid is constructed correctly.

[0136] 2.2 Screening of multi-copy strains

[0137] The high-copy recombinant Pichia pastoris strains were screened, and the screening results were as follows: Figure 8As shown, the results showed that the high-copy recombinant Pichia pastoris strain still had good growth performance under high resistance selection pressure.

[0138] 2.3 PCR identification of positive strains

[0139] Positive colonies were picked for expansion culture, and then the yeast genome was extracted and the bacterial solution was PCR identified. The identification results were as follows: Figure 9 As shown, it can be seen that most colonies have been successfully transformed with the target gene fragment.

[0140] 2.4 Detection of recombinant Pichia pastoris expression products

[0141] After three days of fermentation, the supernatant of the bacterial solution was analyzed by SDS-PAGE electrophoresis. Figure 10-12 The images show the recombinant antimicrobial peptides Gox-LP-pBD1, survivin-LP-pBD1, and PIGF-LP-pBD1. After Coomassie blue staining, distinct protein bands were observed at the predicted sizes (72.1 kDa for Gox-LP-pBD1, 26.6 kDa for survivin-LP-pBD1, and 28.1 kDa for PIGF-LP-pBD1). PIGF-LP-pBD1 showed two distinct protein bands. Compared to the antimicrobial peptide LP-pBD1, the addition of the fusion protein reduced the number of protein bands in the fermentation supernatant, indicating a decrease in protein misfolding, a significant increase in the proportion of correctly folded antimicrobial peptides, a reduction in misfolding, and a significant increase in secretory expression, resulting in significantly enhanced antibacterial activity of the corresponding recombinant antimicrobial peptides.

[0142] IV. Antibacterial Activity Detection of Recombinant Antimicrobial Peptide Protein

[0143] 1. Materials and Methods

[0144] 1.1 Materials

[0145] In this experiment, sodium chloride was purchased from Sinopharm Chemical Reagent Co., Ltd., agar powder was purchased from Beijing Solebow Technology Co., Ltd., tryptone was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and ampicillin was purchased from TheromoFisher.

[0146] P2, P20, P200, and P1000 pipettes: Gilson; MLS-3781L-PC high-pressure steam sterilizer: Panasonic Electric Co., Ltd.; DL-CJ-1NDII clean bench: Beijing Donglian Har Instrument Manufacturing Co., Ltd.; DHP-9052B constant temperature incubator: Shanghai Yiheng Scientific Instrument Co., Ltd.

[0147] 1.2 Experimental methods

[0148] The antibacterial effect of the recombinant antimicrobial peptide protein was determined as follows:

[0149] The antibacterial effect was determined using the Oxford cup method. Single colonies of Escherichia coli, Staphylococcus aureus, and Salmonella were incubated in 5 mL of 2YT medium and incubated at 37°C, 230 rpm, for 12 hours. Afterwards, 20 μL of the bacterial solution was evenly spread on a solid LB medium. Once the bacterial solution was completely absorbed by the medium, five sterilized Oxford cups were placed vertically on the medium, and 200 μL of the test liquid was added to each well. The five wells contained the single-, double-, and triple-concentrated groups (prepared by dissolving the lyophilized powder from Experiment 3 at its original concentration in purified water), a positive control containing 100 μg / mL ampicillin, and the supernatant of the empty vector fermentation broth.

[0150] 2. Results Analysis

[0151] 2.1 Analysis of antibacterial effect

[0152] The results of the Oxford cup antibacterial test of recombinant antimicrobial peptide proteins Gox-LP-pBD1 and survivin-LP-pBD1 are as follows Figure 13 As shown, the lyophilized powder reconstitution solution containing the two recombinant antimicrobial peptide proteins had no significant inhibitory effect on Staphylococcus aureus.

[0153] like Figure 14 As shown, the reconstituted solution of lyophilized powder containing the recombinant antimicrobial peptide protein PIGF-LP-pBD1 exhibited significant inhibitory effects against all three pathogens. The inhibition zones produced by the lyophilized powder against Staphylococcus aureus and Salmonella were significantly larger than those produced against Escherichia coli. This indicates that the lyophilized powder has poor inhibitory effects against E. coli but better inhibitory effects against Staphylococcus aureus and Salmonella. Furthermore, the inhibitory effect of the lyophilized powder increased with increasing lyophilized powder concentration. The inhibitory effect of a triple concentrate of lyophilized powder against Staphylococcus aureus and Salmonella approached that of 100 μg / mL ampicillin.

[0154] The results of the Oxford cup antibacterial test of the recombinant antimicrobial peptide LP-pBD1 are as follows Figure 15 As shown, the empty control group had no inhibitory effect on the three pathogenic bacteria, the ampicillin positive control group had a very significant inhibitory effect on the three pathogenic bacteria, and the lyophilized powder reconstituted solution containing the recombinant antimicrobial peptide protein LP-pBD1 had a small inhibitory effect on the three pathogenic bacteria. The inhibitory effect of the lyophilized powder on Staphylococcus aureus was significantly greater than that on Escherichia coli and Salmonella, and the antibacterial effect of the recombinant antimicrobial peptide protein LP-pBD1 would increase slightly with the increase of the lyophilized powder concentration.

[0155] 2.2 Comparison of antibacterial effects of antimicrobial peptide LP-pBD1 and PIGF-LP-pBD1

[0156] Comparison of the antibacterial activities of three-fold concentrated antimicrobial peptide LP-pBD1 and PIGF-LP-pBD1 Figure 16 As shown, the antimicrobial peptide LP-pBD1 showed weaker antibacterial activity against E. coli, Staphylococcus aureus, and Salmonella than the antimicrobial peptide PIGF-LP-pBD1. Significant differences were observed in the antimicrobial activities of the two antimicrobial peptides. The addition of PIGF significantly improved the correct folding rate, secretion volume, and antimicrobial efficacy of the recombinant antimicrobial peptides. Triple concentrates of both antimicrobial peptides showed the weakest inhibitory activity against E. coli and the strongest inhibitory activity against Staphylococcus aureus.

[0157] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An antibacterial recombinant protein, characterized in that include: (1) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO. 1 and has the same function; or (2) An amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1 and having the same function.

2. The antibacterial recombinant protein according to claim 1, characterized in that It has the amino acid sequence shown in SEQ ID NO.

1.

3. A nucleic acid molecule, characterized in that It comprises a nucleotide sequence encoding the antibacterial recombinant protein according to claim 1 or 2.

4. A carrier molecule, characterized in that It comprises the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 3 or the vector molecule according to claim 4.

6. The method for preparing the antibacterial recombinant protein according to claim 1 or 2, characterized in that: Prepared by artificial synthesis or genetic engineering.

7. An antimicrobial composition, characterized in that The invention comprises the antibacterial recombinant protein according to claim 1 or 2 and auxiliary materials.

8. Use of the antibacterial recombinant protein according to claim 1 or 2 in the preparation of antibacterial products.

9. The use according to claim 8, characterized in that The antimicrobial products include feed, medicine, preservatives, functional foods, biological pesticides, skin care products or antimicrobial coatings.

10. A method for inhibiting bacteria, characterized in that: The method comprises the step of using the antibacterial recombinant protein according to claim 1 or 2.

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