An antibacterial recombinant protein and a preparation method and application thereof

Abstract

The application discloses an antibacterial recombinant protein and a preparation method and application thereof. The antibacterial recombinant protein has excellent bacteriostatic activity, can realize efficient production, and can be used for industrial scale production and preparation. The antibacterial recombinant protein can be used in the fields of feed, medicine, preservative, functional food, biological pesticide, skin care product or antibacterial coating, and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial proteins, and particularly relates to an antibacterial recombinant protein and a preparation method and application thereof. BACKGROUND

[0002] Since the feed industry "banned antibiotics", the research and application of antibacterial peptides as alternative antibiotics have become a hot spot. Pig beta-defensin 1 is an antibacterial peptide found in pigs, which contains three disulfide bonds in its structure, can resist the degradation of protease by forming a rigid structure, and has good inhibitory effect on a variety of bacteria including multi-drug resistant strains, and has certain antiviral activity. Pig beta-defensin 1 can promote the growth of piglets, improve the body condition of sows, and regulate the immune response of pigs. Pig beta-defensin 1 can also participate in affecting various cell signaling pathways, indirectly affecting the process of pig inflammation reaction, etc.

[0003] However, although antibacterial peptides have many advantages, the application of antibacterial peptides still faces severe challenges. On the one hand, the high expression cost of antibacterial peptides limits their large-scale production; on the other hand, the antibacterial effect and safety of antibacterial peptides still need to be further improved. Therefore, the research and development of antibacterial peptide modification and function expansion to optimize the function of antibacterial peptides are very important. SUMMARY

[0004] To solve at least part of the technical problems in the prior art, the present application provides an antibacterial recombinant protein and a preparation method and application thereof. Specifically, the present application includes the following contents.

[0005] In a first aspect of the present application, an antibacterial recombinant protein is provided, comprising:

[0006] (1) an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1 and having the same function; or

[0007] (2) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more than one amino acid of the amino acid sequence shown in SEQ ID NO. 1 and having the same function.

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

[0009] In a second aspect of the present application, a nucleic acid molecule is provided, comprising a nucleotide sequence encoding the antibacterial recombinant protein according to the present application.

[0010] In a third aspect of the present application, a vector molecule is provided, comprising the nucleic acid molecule according to the present application.

[0011] In a fourth aspect of the present application, a host cell comprising the nucleic acid molecule or the vector molecule according to the present application is provided.

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

[0013] In a sixth aspect of the present application, an antibacterial composition comprising the antibacterial recombinant protein according to the present application and an excipient is provided.

[0014] In a seventh aspect of the present application, the use of the antibacterial recombinant protein according to the present application in the preparation of an antibacterial product is provided.

[0015] In certain embodiments, the use according to the present application, wherein the antibacterial product comprises a feed, a drug, a preservative, a functional food, a biopesticide, a skin care product or an antibacterial coating.

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

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

[0018] In a genetic engineering expression system, the activity of an exogenous protein is reduced, cannot be expressed or secreted due to the characteristics of the exogenous gene, protease degradation, protein folding error, protein modification error and the like. In order to realize the normal secretion and expression of the antibacterial peptide in the genetic engineering expression system, an antibacterial recombinant protein is constructed. The antibacterial recombinant protein according to the present application has excellent antibacterial activity and can be used for industrial scale production. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 PCR verification of porcine beta-defensin 1 (pBD1) and LP-pBD1 target gene fragments is shown, wherein the Marker lane is a DNA marker, the first lane is a pBD1 gene, and the second lane is an LP-pBD1 gene.

[0020] Figure 2 SDS-PAGE electrophoresis results of supernatant of Pichia pastoris protein induced expression are shown, wherein the M lane is a protein marker, and the 1, 2 and 3 lanes are supernatants of three strains of Pichia pastoris transformed with pBD1 gene sequence induced for 96h.

[0021] Figure 3SDS-PAGE electrophoresis results of supernatant of Pichia pastoris liquid after protein induction expression are shown, wherein, M is protein marker, 1, 2, 3 are three strains of Pichia pastoris transformed into LP-pBD1 gene sequence supernatant after induction for 96h.

[0022] Figure 4 The pPICZ alpha A plasmid map containing the recombinant antibacterial peptide protein gene PIGF-LP-pBD1 is shown.

[0023] Figure 5 The pPICZ alpha A plasmid enzyme cutting verification results containing the recombinant antibacterial peptide protein gene Gox-LP-pBD1 are shown, wherein, Marker lane is DNA marker, the last two lanes are two groups of pPICZ alpha A plasmids containing recombinant antibacterial peptide protein gene using NotI and EcoRI double enzyme cutting verification.

[0024] Figure 6 The pPICZ alpha A plasmid enzyme cutting verification results containing the recombinant antibacterial peptide protein gene survivin-LP-pBD1 are shown, wherein, Marker lane is DNA marker, the last two lanes are two groups of pPICZ alpha A plasmids containing recombinant antibacterial peptide protein gene using NotI and EcoRI double enzyme cutting verification.

[0025] Figure 7 The pPICZ alpha A plasmid enzyme cutting verification results containing the recombinant antibacterial peptide protein gene PIGF-LP-pBD1 are shown, wherein, Marker lane is DNA marker, the last two lanes are two groups of pPICZ alpha A plasmids containing recombinant antibacterial peptide protein gene using NotI and EcoRI double enzyme cutting verification.

[0026] Figure 8 The YPD medium containing high concentration of Zeocin antibiotic (Zeocin concentration is 2000 μg / mL) is shown to screen the transformed Pichia pastoris cells, wherein, the single colony on the left side is the Pichia pastoris strain containing multiple copies of the target gene.

[0027] Figure 9 The PCR verification of Pichia pastoris strain protein before induction is shown, wherein, Marker lane is DNA marker, 1, 2 lanes are two strains of recombinant antibacterial peptide protein PIGF-LP-pBD1 gene integrated.

[0028] Figure 10The results of SDS-PAGE electrophoresis of the supernatant of the Pichia pastoris liquid after induction of the recombinant antibacterial peptide protein gene Gox-LP-pBD1 are shown. Among them, the Marker lane is the protein marker, and the 1, 2, and 3 lanes are the supernatants of three strains of Pichia pastoris transformed with the Gox-LP-pBD1 gene sequence after 96h induction.

[0029] Figure 11 The results of SDS-PAGE electrophoresis of the supernatant of the Pichia pastoris liquid after induction of the recombinant antibacterial peptide protein gene survivin-LP-pBD1 are shown. Among them, the Marker lane is the protein marker, and the 1, 2, and 3 lanes are the supernatants of three strains of Pichia pastoris transformed with the survivin-LP-pBD1 gene sequence after 96h induction.

[0030] Figure 12 The results of SDS-PAGE electrophoresis of the supernatant of the Pichia pastoris liquid after induction of the recombinant antibacterial peptide protein gene PIGF-LP-pBD1 are shown. Among them, the Marker lane is the protein marker, and the 1, 2, and 3 lanes are the supernatants of three strains of Pichia pastoris transformed with the PIGF-LP-pBD1 gene sequence after 96h induction.

[0031] Figure 13 The results of the antibacterial effect of the freeze-dried powder of the supernatant of the Pichia pastoris liquid after 96h induction of the recombinant antibacterial peptide protein genes Gox-LP-pBD1 and survivin-LP-pBD1 are shown. The sample wells marked "1, 2, and 3" are the one-fold, two-fold, and three-fold concentrated groups prepared by dissolving the freeze-dried powder in pure water at the original concentration. The sample well marked "A" is the positive control group containing 100μg / mL ampicillin. The left side is the antibacterial effect diagram of the recombinant protein Gox-LP-pBD1, and the right side is the antibacterial effect diagram of the recombinant protein survivin-LP-pBD1.

[0032] Figure 14 The results of the antibacterial effect of the freeze-dried powder of the supernatant of the Pichia pastoris liquid after 96h induction of the recombinant antibacterial peptide protein gene PIGF-LP-pBD1 are shown. The sample wells marked "1x, 2x, and 3x" are the one-fold, two-fold, and three-fold concentrated groups prepared by dissolving the freeze-dried powder in pure water at the original concentration. The sample well marked "A" is the positive control group containing 100μg / mL ampicillin. The sample well marked "Culture" is the supernatant of the empty vector fermentation liquid.

[0033] Figure 15The bacteriostatic results of the freeze-dried powder of the supernatant of the Pichia pastoris into which the recombinant antibacterial peptide protein gene LP-pBD1 is introduced after 96h of induction are shown, wherein the sample wells marked as "1x, 2x, 3x" are one-time, two-time and three-time concentrated groups prepared by dissolving the freeze-dried powder in pure water at the original concentration, the sample well marked as "A" is a positive control group containing 100 μg / mL ampicillin, and the sample well marked as "culture" is the supernatant of the fermentation liquid of the empty vector.

[0034] Figure 16 The bacteriostatic effects of the recombinant antibacterial protein LP-pBD1 and PIGF-LP-pBD1 are shown, wherein the comparative sample is a three-time concentrated recombinant antibacterial protein LP-pBD1 and PIGF-LP-pBD1 fermentation liquid supernatant freeze-dried powder reconstituted solution, and the figure from left to right is E. coli, S. aureus and S. enterica. DETAILED DESCRIPTION

[0035] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0036] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it is understood that the upper limit and the lower limit of the range and every intermediate value between them are specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the content of this specification and that of any document incorporated herein by reference, the content of this specification prevails.

[0038] Antibacterial recombinant proteins

[0039] In one aspect of the present application, an antibacterial recombinant protein is provided, comprising:

[0040] (1) an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1 and having the same function; or

[0041] (2) the amino acid sequence shown in SEQ ID NO. 1 modified, substituted, deleted or added with one or more than one amino acid 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 application, "antibacterial" refers to inhibiting the growth or activity of bacteria, inhibiting the proliferation of bacteria or directly killing bacteria. The bacteria include gram-positive bacteria and gram-negative bacteria. 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 israelii), Mycobacterium (such as, but not limited to, Mycobacterium tuberculosis, Mycobacterium leprae, non-tuberculous Mycobacterium, etc.), Erysipelothrix (such as, but not limited to, Erysipelothrix rhusiopathiae), 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, etc.

[0044] In the present application, 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 application. To determine sequence identity, sequence alignment can be performed, which can be done in various 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 are able to determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment of the full-length sequences being compared.

[0045] In the present application, the sequence of the antibacterial recombinant protein obtained by modifying, substituting, deleting or adding one or more than one amino acid also belongs to the protection scope of the present application. The term "modifying" refers to any chemical modification of the amino acid sequence. The term "substituting" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to the naturally occurring molecule.

[0046] In the present application, the detection method of antibacterial effect is not particularly limited, which can be carried out by the methods known in the art, such as Oxford cup method, filter paper method, inhibition zone method, minimum inhibitory concentration determination, time-kill curve method, etc.

[0047] In the genetic engineering expression system, the activity of antibacterial peptide pig β-defensin 1 is reduced, cannot be expressed or secreted due to the characteristics of exogenous genes, protease degradation, protein folding error, protein modification error, etc. In order to realize the normal secretion and expression of β-defensin 1 in the genetic engineering expression system, the present application constructs a short antibacterial peptide with linear structure, i.e. linker peptide (LP). The construction of linker peptide to the N-terminal of β-defensin 1 can ensure the correct folding of the protein structure of β-defensin 1 while enhancing the antibacterial activity of β-defensin 1. However, after being connected with the linker peptide, the antibacterial activity of antibacterial peptide pig β-defensin 1 is still weak, and the expression amount is low. Therefore, through a large number of screening experiments, the present application attempts to connect different proteins with antibacterial peptides to improve the activity of antibacterial peptide proteins. The present application studies the simultaneous connection of glucose oxidase (Gox), survivin, placental growth factor (PIGF) and linker peptide to the front end of β-defensin 1, and the results show that glucose oxidase and survivin have no improvement effect on the activity of antibacterial peptide, while connecting placental growth factor to the front end of the recombinant antibacterial peptide can greatly improve the antibacterial activity of antibacterial peptide pig β-defensin 1, increase the expression amount, and thus be suitable for industrial scale production and preparation.

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

[0049] Nucleic acid molecules

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

[0051] The nucleic acid molecule of the present application comprises a codon-optimized coding sequence. A codon refers to a set of every three adjacent nucleotides in a messenger RNA molecule, which represents a certain amino acid in the process of protein synthesis. "Codon optimization" is intended to include the alteration of the codon composition of a recombinant nucleic acid without changing the amino acid sequence.

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

[0053] Once the coding sequence of the antibacterial recombinant protein according to the present application is obtained, the antibacterial recombinant protein can be obtained in large quantities using a recombinant technique. An exemplary method is to clone the coding gene thereof into a vector, and then to transfer into a cell, and then to isolate from the proliferated host cell by a conventional method.

[0054] Vector molecules

[0055] In an aspect of the present application, there is provided a vector molecule comprising the nucleic acid molecule according to the present application.

[0056] The vector of the present application refers to an artificial construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. The vector of the present application is not limited, and can be an expression vector, a viral vector, and the like. In certain embodiments, the vector comprises a gene of interest encoding the antibacterial recombinant protein of the present application, a promoter, a terminator, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. The known vector includes a plasmid vector, a lentivirus vector, an adenovirus vector, an AAV virus vector, and the like.

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

[0058] Host cells

[0059] In an aspect of the present application, there is provided a host cell comprising the nucleic acid molecule according to the present application or the vector molecule according to the present application.

[0060] The host cell of the present application refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising the nucleic acid molecule of the present application. The host cell includes any progeny of the parent cell which is not identical to the parent cell due to mutations that occur during replication.

[0061] Methods of manufacture

[0062] In one aspect of the present application, a method for preparing the antibacterial recombinant protein of the present application is provided. The method for preparation is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0063] In certain embodiments, the antibacterial recombinant protein of the present application is obtained by artificial synthesis. Methods for artificial synthesis of the antibacterial recombinant protein are known in the art, for example, the antibacterial recombinant protein of the present application is obtained by direct synthesis of amino acids.

[0064] In certain embodiments, the antibacterial recombinant protein of the present application is obtained by genetic engineering expression. Genetic engineering expression systems 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 the prokaryotic cell expression system include, but are not limited to, Escherichia coli expression systems. The eukaryotic cell expression system includes, but is not limited to, yeast expression systems, insect cell expression systems, mammalian cell expression systems, etc.

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

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

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

[0068] (3) collecting the antibacterial recombinant protein and purifying.

[0069] Antibacterial compositions

[0070] In one aspect of the present application, an antibacterial composition comprising the antibacterial recombinant protein according to the present application and an adjuvant is provided. The adjuvant can be any suitable carrier or adjuvant used for producing or preparing feed, medicine, preservative, functional food, biopesticide, skin care product, or antibacterial coating, examples of which include, but are not limited to, at least one of diluent, filler, absorbent, wetting agent, binder, preservative, and antioxidant. Among them, examples of the diluent include, but are not limited to, physiological saline, aqueous buffer solution, solvent, dispersion medium, etc.; the filler includes, but is not limited to, starch, lactose, mannitol, microcrystalline cellulose, etc.; the absorbent includes, but is not limited to, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent includes, but is not limited to, water, ethanol, etc.; the binder includes, but is not limited to, hydroxypropyl methyl cellulose, povidone, microcrystalline cellulose, etc.; the preservative includes, but is not limited to, nipagin, chlorobutanol, phenol, sorbic acid, etc.; the antioxidant includes, but is not limited to, ascorbic acid, methionine, etc.

[0071] Uses

[0072] In one aspect of the present application, the use of the antibacterial recombinant protein according to the present application in the preparation of an antibacterial product is provided.

[0073] In the present application, examples of the antibacterial product include, but are not limited to, feed, medicine, preservative, functional food, biopesticide, skin care product, antibacterial coating, etc.

[0074] Methods of inhibiting bacteria

[0075] In one aspect of the present application, a method for inhibiting bacteria is provided, which comprises the step of using the antibacterial recombinant protein according to the present application. The application fields thereof include, but are not limited to, equipment and environmental disinfection, antibacterial material preparation, food processing, agricultural planting and breeding, experimental research, etc.

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

[0077] In the present application, "inhibiting bacteria", "bacteriostasis", and "antibacterial" are used interchangeably, which means inhibiting the growth or activity of bacteria, inhibiting the proliferation of bacteria, or directly killing bacteria.

[0078] Examples

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

[0080] I. Expression and identification of porcine beta defensin pBD1

[0081] 1. Materials and methods

[0082] 1.1 Materials and instruments

[0083] The plasmid used in this example, X33 Pichia pastoris strain, and Escherichia coli, Staphylococcus aureus, Salmonella strains are from the laboratory of Dong Na, College of Animal Science and Technology, China Agricultural University.

[0084] Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, methanol, glucose, etc. were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Carrier DNA, X33 competent cells, lithium acetate, Zeocin antibiotic were purchased from Beijing Coolab Technology Co., Ltd. Biotin, YNB, sorbitol, agar, etc. were purchased from Beijing Solabio Technology Co., Ltd. PCR Mix, protein Marker, etc. were purchased from Yixing Biological Technology Co., Ltd. Related primers were synthesized by Beijing Ruibo Xingke Co., Ltd. Yeast extract, peptone, etc. were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Tool enzymes NotI, EcoRI, etc. were purchased from NEB company.

[0085] ZQZY-88CES, ZQZYB8ES, ZQZYC8ES type constant temperature oscillation incubator: Shanghai Zhichu Instrument Co., Ltd.; DHP-9052B type constant temperature incubator: Shanghai Yiheng Scientific Instrument Co., Ltd.; CWE811 type PCR instrument: Beijing Kangwei Century Biological Technology Co., Ltd.; PowerPac300 type electrophoresis instrument: Bio-Rad; TANON-1600 type gel imaging system: Shanghai Tian Neng Technology Co., Ltd.; 5424 type table centrifuge: Eppendorf; NanoDrop Lite type spectrophotometer: ThermoFisher; MLS-3781L-PC type high-pressure steam sterilization pot: Panasonic Electric Co., Ltd.; P2, P20, P200, P1000 type pipette: Gilson; DL-CJ-1NDII type super clean bench: Beijing Donglian Haer Instrument Manufacturing Company.

[0086] 1.2 Experimental method

[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 Company, and the fragment was spliced into the pPICZαA plasmid vector to construct the recombinant expression plasmid pPICZαA-pBD1.

[0089] The company synthesized recombinant plasmid pPICZαA-pBD1 was transformed into E. coli DH5α competent cells, and the transformed E. coli competent cells were plated on 2YT solid medium containing Zeocin antibiotic and incubated for 12 h. A single E. coli colony of appropriate size was picked and inoculated into 5 mL of 2YT medium containing Zeocin 100 μg / mL, and incubated at 37°C, 220 rpm for 12 h for plasmid extraction. The recombinant plasmid was digested with EcoRI and NotI, and the reaction system is shown in Table 1. The reaction was carried out at 37°C for 2 h, and 10 μL was subjected to 1% agarose gel electrophoresis. The recombinant plasmid was sequenced, and the sequence obtained by sequencing was compared with the optimized sequence by Snapgene software. The plasmid with correct sequencing and enzyme digestion results was selected for transformation.

[0090] 1.2.2 Recombinant antibacterial protein expression and identification in Pichia pastoris

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

[0092] Table 1 Linearization system of recombinant plasmid

[0093]

[0094] Take 30 μL of linearized plasmid and 10 μL of pre-denatured Carrier DNA, add to 200 μL of non-melted competent cells, and place the mixed system in a 30°C water bath. Mix every 15 s until the competent cells just melt. Add 1.4 mL of B2 solution to the system and mix, and incubate in a 30°C water bath for 60 min, mixing every 20 min. Centrifuge at 3000 rpm for 3 min, discard the supernatant, and resuspend the bacterial pellet in 1 mL of B3 solution. Centrifuge at 3000 rpm for 3 min, discard the supernatant, and resuspend the bacterial pellet in 1 μL of B3 solution. Spread 100 μL of bacterial solution onto YPD medium with Zeocin resistance, and incubate at 30°C for 3 days until yeast colonies grow on the YPD medium.

[0095] Multiple single colonies were picked from YPD medium containing successfully transformed yeast into 100 mL conical flask containing 10 mL YPD liquid medium with 100 μg / mL Zeocin antibiotic, and cultured at 28°C, 220 rpm for 24 h. Then 100 μL of the bacterial solution was taken into a 1.5 mL centrifuge tube, and the centrifuge tube was placed in liquid nitrogen for 5 min, then transferred to a 95°C metal bath for heating for 5 min, and then transferred to liquid nitrogen for freezing again. This was repeated three times, and then PCR identification was performed using the repeatedly frozen and thawed bacterial solution. 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] The identified single colonies were inoculated into 10 mL YPD liquid medium and cultured at 28°C, 220 rpm for 24 h. Then 100 μL of the bacterial solution was inoculated into 50 mL BMGY medium and cultured until the OD600 of the bacterial solution reached 2.0. The bacterial solution was placed in a 50 mL centrifuge tube and centrifuged at 4000 rpm, 4°C for 15 min. The supernatant was discarded and the bacterial pellet was resuspended with BMMY medium. The resuspended bacterial solution was transferred into 50 mL BMMY medium for induction, and methanol was added every 12 h. The initial methanol concentration in the culture medium was 0.5%, and then the amount of methanol added was increased by 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1% successively. The supernatant was taken every 24 h, 5x loading buffer was added to the sample, and the sample was heated in a 95°C metal bath for 40 min. Then the sample was subjected to 12% SDS-PAGE electrophoresis experiment, and the protein content in the supernatant was detected.

[0102] 2. Results analysis

[0103] 2.1 PCR identification of target gene fragment

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

[0105] 2.2 Detection of Pichia pastoris expression product

[0106] After three days of fermentation, the supernatant of the bacterial solution was subjected to SDS-PAGE electrophoresis analysis, as shown in Figure 2 the Coomassie brilliant blue staining and destaining, no obvious band was observed at the expected protein size position. The antibacterial peptide pBD1 failed to be successfully expressed and secreted.

[0107] II. Expression and identification of recombinant antibacterial peptide LP-pBD1

[0108] 1. Materials and methods

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

[0110] 1.2 Experimental methods

[0111] Based on the amino acid sequence of pig beta defensin pBD1 in Experiment One, a linker peptide LP was added to the N-terminus of pBD1 to splice into recombinant antibacterial peptide LP-pBD1. The codon optimization website was used to optimize the LP-pBD1 gene sequence to adapt to the expression preference of Pichia pastoris. The gene fragment was synthesized by Beijing Ruibo Xingke Company, and the fragment was spliced into the pPICZ alpha A plasmid vector to construct the recombinant expression plasmid pPICZ alpha A-LP-pBD1.

[0112] Table 4 LP amino acid sequence

[0113]

[0114] The recombinant plasmid was obtained and identified, and the method of Pichia pastoris expression and identification of recombinant antibacterial peptide protein was the same as Experiment One.

[0115] 2. Results analysis

[0116] 2.1 PCR identification of target gene fragment

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

[0118] 2.2 Detection of Pichia pastoris expression products

[0119] After three days of induction fermentation, the supernatant of the bacterial solution was subjected to SDS-PAGE electrophoresis analysis, as shown in Figure 3As shown, after Coomassie brilliant blue staining and destaining with destaining solution, there was an obvious band at about 8.1 kDa, indicating that the recombinant antibacterial peptide LP-pBD1 was successfully expressed and secreted in Pichia pastoris. There were three obvious bands of protein in the supernatant, which was speculated to be due to the characteristics of disulfide bond. Some disulfide bonds of antibacterial peptides were destroyed by reductase in the endoplasmic reticulum of Pichia pastoris, thereby forming misfolded. However, most of the antibacterial peptides formed stable disulfide bonds, were correctly folded to form stable structures, and had antibacterial activity. The recombinant antibacterial peptide LP-pBD1 showed a small amount of antibacterial activity in subsequent verification.

[0120] III. Expression and identification of recombinant antibacterial peptides Gox-LP-pBD1, survivin-LP-pBD1 and PI GF-LP-pBD1

[0121] 1. Materials and methods

[0122] 1.1 Materials and experimental methods

[0123] 1.2 Experimental methods

[0124] 1.2.1 Construction and identification of expression vector

[0125] Based on the recombinant antibacterial peptide amino acid sequence of the expression and identification of recombinant antibacterial peptide LP-pBD1 in Experiment Two, three different proteins, glucose oxidase Gox, survivin and growth factor PI GF, were added to the N-terminus of the recombinant antibacterial peptide to splice into recombinant antibacterial peptides Gox-LP-pBD1, survivin-LP-pBD1 and PI GF-LP-pBD1. The codon optimization website was used to optimize the gene sequences of Gox-LP-pBD1, survivin-LP-pBD1 and PI GF-LP-pBD1 to adapt to the expression preference of Pichia pastoris. The gene fragment was synthesized by Beijing Ruiboxingke Company, and the fragment was spliced into the pPICZαA plasmid vector to construct the recombinant expression plasmids pPICZαA-Gox-LP-pBD1, pPICZαA-survivin-LP-pBD1 and pPICZαA-PI GF-LP-pBD1. Figure 4 The pPICZαA plasmid containing the recombinant antibacterial peptide protein gene PI GF-LP-pBD1 is shown. This plasmid is the final practical plasmid.

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

[0127]

[0128]

[0129] 2.2.1 Recombinant plasmid acquisition and identification, Pichia pastoris expression and identification method of recombinant antibacterial peptide protein

[0130] 1.2.2 High copy Pichia pastoris screening and Pichia pastoris flask induction expression

[0131] The identified yeast was inoculated into a high concentration of Zeocin (Zeocin content was 2000 μg / mL) and cultured for four days. After that, the well-grown colonies were picked and streaked for preservation. A single colony was picked from the YPD plate of the high copy yeast and inoculated into 10 mL of YPD liquid medium, and the bacteria were shaken at 28°C and 220 rpm for 24 h. Then 100 μL of the bacterial solution was inoculated into 50 mL of BMMY medium, and the bacterial solution was placed in a 50 mL centrifuge tube and centrifuged at 4000 rpm and 4°C for 15 min. The supernatant was discarded, and the bacterial pellet was resuspended with BMMY medium and transferred to 50 mL of BMMY medium for induction. Methanol was added every 12 h, and the initial methanol concentration in the culture medium was 0.5%. Then the amount of methanol added was increased by 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1% each time. The supernatant was taken every 24 h, 5x loading buffer was added to the sample, and the sample was heated in a 95°C metal bath for 40 min. Then the sample was subjected to 12% SDS-PAGE electrophoresis experiment, and the protein content in the supernatant was detected.

[0132] The fermentation broth was divided into centrifuge tubes, and the broth was centrifuged at 4°C and 4000 rpm for 1 h. The supernatant was discarded, and the freeze-drying machine was used 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 results of double enzyme digestion of the constructed recombinant expression plasmid 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 shown in Figures 5-7 The sequencing results also showed that the plasmid construction was correct.

[0136] 2.2 Screening of multiple copy strains

[0137] The high copy recombinant Pichia pastoris strains were screened, and the screening results were as shown in Figure 8As shown in the results, the high copy recombinant Pichia pastoris strains still have good growth performance under high resistance screening 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 liquid was PCR identified, and the identification results are shown in Figure 9 As can be seen, most colonies have successfully transformed into 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 liquid was analyzed by SDS-PAGE electrophoresis, Figures 10-12 As shown in the results, the high copy recombinant Pichia pastoris strains still have good growth performance under high resistance screening pressure.

[0142] Four, detection of antibacterial activity of recombinant antibacterial peptide protein

[0143] 1. Materials and methods

[0144] 1.1 Materials

[0145] In this experiment, sodium chloride was purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd., agar powder was purchased from Beijing Solaybao Technology Co., Ltd., tryptone was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and ampicillin was purchased from TheromoFisher company.

[0146] P2, P20, P200, P1000 type pipette: Gilson; MLS-3781L-PC type high pressure steam sterilization pot: Matsushita Electric Co., Ltd.; DL-CJ-1NDII type super clean bench: Beijing Donglian Haer instrument manufacturing company; DHP-9052B type constant temperature incubator: Shanghai Yiheng Scientific Instrument Co., Ltd.

[0147] 1.2 Experimental method

[0148] The antibacterial effect of recombinant antimicrobial peptides 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 picked and incubated in 5 mL of 2YT medium at 37°C and 230 rpm for 12 hours. Then, 20 μL of the bacterial suspension was evenly spread onto LB solid medium. After the bacterial suspension was completely absorbed by the medium, five sterilized Oxford cups were placed vertically on the medium. 200 μL of the test liquid was added to each well. The five wells represented the following groups: a one-fold concentrated group, a two-fold concentrated group, a three-fold concentrated group (prepared by dissolving the lyophilized powder at its original concentration in purified water), a positive control group containing 100 μg / mL ampicillin, and the supernatant of the empty carrier fermentation broth.

[0150] 2. Results Analysis

[0151] 2.1 Analysis of antibacterial effect

[0152] Results of Oxford Cup antibacterial assays for recombinant antimicrobial peptides Gox-LP-pBD1 and survivin-LP-pBD1 are as follows: Figure 13 As shown, the lyophilized powder reconstituted solution containing two recombinant antimicrobial peptides had no significant inhibitory effect on Staphylococcus aureus.

[0153] like Figure 14 As shown, the reconstituted solution of lyophilized powder containing recombinant antimicrobial peptide PIGF-LP-pBD1 significantly inhibited the growth of three pathogenic bacteria. The inhibition zones produced by the lyophilized powder in Staphylococcus aureus and Salmonella were significantly larger than those in Escherichia coli. This indicates that the lyophilized powder had a poor inhibitory effect on Escherichia coli, but a better inhibitory effect on Staphylococcus aureus and Salmonella. Furthermore, the inhibitory effect of the lyophilized powder increased with increasing concentration; the three-fold concentrated solution of the lyophilized powder showed an inhibitory effect close to that of ampicillin in Staphylococcus aureus and Salmonella.

[0154] Results of the Oxford cup antibacterial assay for 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, while the ampicillin positive control group had a highly significant inhibitory effect on all three pathogenic bacteria. The lyophilized powder reconstituted solution containing recombinant antimicrobial peptide protein LP-pBD1 had a slight 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 recombinant antimicrobial peptide protein LP-pBD1 increased slightly with the increase of lyophilized powder concentration.

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

[0156] Comparison of antibacterial activity of three-fold concentrated antibacterial peptide LP-pBD1 and PIGF-LP-pBD1 Figure 16 As shown in the figure, the antibacterial effect of antibacterial peptide LP-pBD1 on E. coli, S. aureus and Salmonella is weaker than that of antibacterial peptide PIGF-LP-pBD1, and there is a significant difference in the antibacterial activity of the two antibacterial peptides. The correct folding rate, secretion amount and antibacterial effect of the recombinant antibacterial peptide are significantly improved after the addition of PIGF. The three-fold concentrated solution of the two antibacterial peptides has the weakest inhibition on E. coli and the strongest inhibition on S. aureus.

[0157] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can still be modified, or some technical features can be replaced by equivalents. The modification or replacement does not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An antibacterial recombinant protein, characterized in that, The amino acid sequence of the antibacterial recombinant protein is shown as SEQ ID NO.

1.

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

3. A carrier molecule characterized in that, It comprises the nucleic acid molecule according to claim 2.

4. A host cell characterized in that, It comprises the nucleic acid molecule according to claim 2 or the vector molecule according to claim 3.

5. The method for preparing the antimicrobial recombinant protein according to claim 1, characterized in that, It is prepared by artificial synthesis or genetic engineering.

6. An antibacterial composition characterized by, It comprises the antibacterial recombinant protein according to claim 1 and adjuvants.

7. Use of the antibacterial recombinant protein according to claim 1 for the preparation of an antibacterial product, characterized in that, The antibacterial is against Escherichia coli, Staphylococcus aureus or Salmonella.

8. Use according to claim 7, characterized in that, The antibacterial product comprises a drug, a preservative, a biological pesticide, a skin care product or an antibacterial coating.

9. A method of inhibiting bacteria for non-therapeutic use, characterized in that, It comprises the step of using the antibacterial recombinant protein according to claim 1, and the bacteria are Escherichia coli, Staphylococcus aureus or Salmonella.

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