KR20110121852A - Method for producing antimicrobial peptide and use of the same - Google Patents

Abstract

PURPOSE: A method for preparing peptides having antibacterial activity is provided to produce a large amount of beta-defensin. CONSTITUTION: A method for in vitro expression of pig beta-defensin comprises: a step of performing PCR using pig tissue-derived cDNA as a template and primer for amplifying beta-defensin; a step of treating restriction enzyme to the PCR product; a step of performing ligation of the PCR product; and a step of transforming an expression vector to a host cell. The primer has a base sequence of sequence number 1 or 2. The host cell is yeast. An antibacterial composition against pathogenic microorganism contains beta-defensin. The pathogenic microorganism is gram-positive such as Bacillus cereus or Bacillus cereus or gram-negative bacteria such as E.coli or Salmonella enteritidis.

Description

Method for producing antibiotic peptide and application of the peptide {Method for producing antimicrobial peptide and use of the same}

The present invention relates to a method for producing a peptide having antimicrobial activity and to the use of a peptide produced through the method.

The emergence of bacterial strains resistant to conventional antibiotics has prompted the study of new therapeutic agents, including antimicrobial peptides of animal origin. Antimicrobial peptides are recognized to play an important role in the inherent host defense mechanisms of most living organisms, including plants, insects, amphibians and mammalian organisms, and possess potential antimicrobial activity against bacteria, fungi and certain viruses. It is known.

Antimicrobial peptides are low molecular weight molecules of less than 5 kilodaltons that possess broad-spectrum activity and constitute an important part of host defense against microbial infection, providing a starting point for designing low molecular weight antimicrobial compounds.

Most antimicrobial peptides do not target specific molecular receptors of the pathogen but rather affect and penetrate the microbial membrane. They are known to have a tendency to fold into both hydrophilic structures with clusters of hydrophobic and charged regions, which are properties contributing to their membrane activity. Antimicrobial peptides easily distinguish more than 95% of peptides incorporated into lipids to compromise membrane integrity into phospholipid bilayers.

In bacteria, antimicrobial peptides can lead to a small temporary increase in conductivity in planar lipid bilayers, thereby partially canceling out the polarity of the cellular membrane potential gradient. As bacteria greatly develop antibiotic resistance, the potential for the development of antimicrobial peptides becomes clear as novel therapeutics that can overcome the problem of antibiotic resistance.

The protective function of antimicrobial peptides in their own host defense mechanisms has been demonstrated in Drosophilia, where reduced expression of these peptides dramatically reduced survival after microbial attack. In mammals, a similar function is suggested due to incomplete bacterial killing in the lungs and small mice in patients with cystic fibrosis.

Currently, the properties of hundreds of antimicrobial peptides have been described. Generally, naturally occurring antimicrobial peptides are 12-50 amino acids in length and are divided into several structural groups, including α-helix, β-sheets, extended peptides and looped peptides.

Although these peptides exhibit significant diversity, they have two general and functionally important requirements: pure cationic, which facilitates interaction with the negatively charged microbial surface, and an amphiphilic that allows integration into the microbial membrane. The ability to assume a fascination structure.

Antimicrobial peptides found in mammals are classified into cysteine-defensin (α- and β-defensins) and cathelicidins.

In addition, while breeding cattle, pigs, chickens, etc., antibiotics for animals are mixed and fed into the feed, which causes antibiotic-resistant bacteria in the animals, which are transferred to people who eat livestock. According to the Food and Drug Administration's Monitoring of Antibiotic Resistance of Food Poisoning Bacteria for 7 months from April 2005, food-resistant antibiotic-resistant bacteria are also of concern, as E. coli detected in meat has a 92.5% resistance to antibiotics. Appeared to be. Currently, the development of antibiotic substitutes is in progress, but apart from this, there is a need for the development of a technology using bacterial defense materials naturally generated in vivo.

Antibiotics can be added to pig feed to improve the growth rate and feed efficiency by inhibiting the growth of harmful microorganisms, increasing the utilization and absorption rate of nutrients, and preventing diseases by increasing immunity and therapeutic effects by participating in intestinal metabolism. . However, by abuse of antibiotics, there is a difficulty in treating animals due to the induction of antibiotic resistant bacteria, and the resistant bacteria remaining in the livestock products can be transferred to humans, causing shock resistant bacteria, physical development abnormalities, and the like. Thus, international organizations such as the World Health Organization (WHO) have begun to regulate antibiotics. Most of the antimicrobial effects developed previously (eg yolk sulphate, probiotics, organic acids, Mannanoligosaccharides, Fructooligosaccharides, chitosan, etc.) have been demonstrated at the in-vitro level, and the relationship with the mucosal immune system is insufficient. In addition, as a result of animal experiments, the antimicrobial effect is ambiguous and unlimited use due to the increase in production cost is difficult.

However, one of the innate immunity reactants, which are in vivo molecules called antimicrobial peptides, the natural antibiotic defensin, regardless of the type of microorganism, can act immediately or within hours. have. There is no problem of resistant bacteria, a problem of conventional antibiotics, and there are no side effects or rejection problems for acquired immunity. In addition, when a problem occurs in the expression or regulation mechanism of defensin, various infectious diseases are caused, and it is predicted that different defensins work better in different bacterial groups. Thus, defensin research is of great industrial importance. Defensins serve as a bridge between innate immunity and acquired immunity as well as innate immunity, and human defensins have been shown to have antiviral effects such as antimicrobial activity and AIDS suppression ability. Defensins have several hundred times stronger potency than the antimicrobial activity of lysozyme, and human defensins not only act specifically on pathogens, but also vary in the range of treatment due to their synergism. There is a possibility of establishing a new infection treatment method through induction expression, and there is a possibility of industrialization as a feed additive by mass production by genetic manipulation.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide an in vitro expression system of defensin.

Another object of the present invention is to provide a pharmaceutical composition comprising defensin obtained from the in vitro expression system.

Still another object of the present invention is to provide a feed composition containing defensin obtained from the in vitro expression system.

In order to achieve the above object, the present invention comprises the steps of: a) performing PCR using primers for amplifying beta-diffsin using cDNA derived from porcine tissue as a template; b) subjecting the PCR product to restriction enzymes; c) ligating the restriction enzyme-treated PCR product to the expression vector of FIG. 2; And d) provides a method for in vitro expression of porcine beta defensin comprising the step of transforming the expression vector into a host cell.

In one embodiment of the invention, the primer preferably has a base sequence selected from the group consisting of SEQ ID NO: 1 and 2, but is not limited thereto.

In one embodiment of the present invention, the beta-defensin preferably has an amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

In addition, the present invention comprises the steps of a) PCR using a primer for amplifying beta-diffsin using a cDNA derived from pig tissue as a template; b) subjecting the PCR product to restriction enzymes; c) constructing the expression vector of FIG. 6 by ligating the restriction enzyme-treated PCR product to an expression vector; And d) provides a method for in vitro expression of porcine beta defensin comprising the step of transforming the expression vector into a host cell.

In one embodiment of the present invention, the primer preferably has a base sequence selected from the group consisting of SEQ ID NO: 3 and 4, but is not limited thereto.

In another embodiment of the present invention, the host cell is preferably yeast, but is not limited thereto.

In another aspect, the present invention provides a composition for antimicrobial against pathogenic microorganisms, including beta-defensin prepared by the expression method of the present invention.

In one embodiment of the present invention, the pathogenic microorganism is preferably Gram-positive or Gram-negative bacteria, and the Gram-positive bacteria is Bacillus cereus or Staphylococcus aureus, and the Gram-positive bacteria is Escherichia coli or Salmonella enteritidis. More preferably, but is not limited thereto.

The present invention is to build a yeast strain expression vector (Fig. 6) to test the antimicrobial efficacy of the expression product (lysate or isolation or purified products) through yeast cytoplasmic expression of porcine beta defensin, into the yeast strain Introducing and confirming the antimicrobial function of the final product through the culture of the introduced yeast strain (pGAL-10 vector is a vector specifically expressed in yeast).

In order to test the antimicrobial efficacy of pig beta defensin 1,2,3 expression products, the expression of yeast cytoplasm is induced using only the mature peptide sequence in each beta defensin. Accordingly, after confirming each mature peptide structure, it is inserted into the yeast expression vector and introduced into the yeast to induce expression. To identify each mature peptide for expression in yeast, check the information of the NCBI gene bank and identify cDNA and sequence in each mRNA for it. However, for the expression of E. coli or yeast, not the expression of animal cells, it is necessary to confirm the size of the final peptide except for the transcript factor. In particular, the mature peptide sequence is identified by comparing the beta defensin gene structure of pigs and humans.

Before discussing the present invention in more detail, the following terms and conventions are first defined.

The term "antibacterial activity" is defined herein as an activity capable of killing or inhibiting the growth of microbial cells. In the context of the present invention, the term "antibacterial" means bactericidal and / or bacteriostatic and / or fungicidal and / or bacteriostatic effect and / or viral bactericidal effect, wherein the term "sterilization" is intended to kill bacterial cells. It must be understood. The term "bacterial" should be understood as being able to inhibit bacterial growth, ie, inhibit the growth of bacterial cells. The term "fungicidal" should be understood as capable of killing fungal cells. The term “fungal fungus” should be understood as being able to inhibit fungal growth, ie, inhibit the growth of fungal cells.

The term "virus sterilization" should be understood as being able to inactivate viruses. The term "microbial cell" refers to a bacterial or fungal cell (including yeast).

For the purposes of the present invention, the antimicrobial activity can be determined according to the procedure described in the Journal of Immunological Methods (137 (2), pp. 167-174 (1991)) by Lehrer et al.

As used in this context, the term “cDNA” includes DNA molecules that can be prepared by reverse transcription from mature spliced mRNA molecules derived from eukaryotic cells. cDNA lacks the intron sequence normally present in the corresponding genomic DNA. Early primary RNA transcripts are precursors of mRNA, which undergo a series of processing events before appearing as mature spliced mRNA. These events include the removal of intron sequences by a process named splicing. Therefore, when cDNA is derived from mRNA, it lacks the intron sequence.

As used herein, the term “nucleic acid construct” refers to a single- or double-stranded nucleic acid molecule that has been isolated from a naturally occurring gene or modified to contain segments of nucleic acid in a manner that does not exist in nature. When the nucleic acid construct contains a control sequence necessary for the expression of the coding sequence of the present invention, the term "nucleic acid construct" is synonymous with the term "expression cassette".

The term "control sequence" is defined herein to include all components necessary or advantageous for expression of a polypeptide of the invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At least the control sequence includes a promoter and a transcriptional and translational disruption signal. Control sequences may be provided with a linker for the purpose of introducing specific restriction sites that facilitate the ligation of the coding region of the control sequence and the nucleotide sequence encoding the polypeptide.

The term “operably linked” is defined herein as a conformation such that the control sequence is appropriately positioned at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of the polypeptide.

As used herein, the term “coding sequence” includes nucleotide sequences that directly specify the amino acid sequence of the protein product. The boundaries of the coding sequences are usually determined by an open reading frame that usually begins with an ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

In this context, the term “expression” includes any step involved in the production of a peptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

In this context, the term “expression vector” includes a linear or cyclic DNA molecule comprising a segment encoding a peptide of the invention and operably linked to additional segments that provide for its transcription.

The term "host cell" as used herein includes any cell type that accepts transformation with nucleic acid constructs.

Hereinafter, the present invention will be described.

The present invention relates to nucleic acid constructs that produce beta defensin comprising a nucleotide sequence of the invention operably linked to one or more control sequences that direct expression of a coding sequence in a suitable host cell under conditions compatible with the control sequence.

Nucleotide sequences encoding peptides of the invention can be engineered in a variety of ways to provide expression of the peptides. Depending on the expression vector, nucleotide sequence manipulation prior to insertion into the vector may be desirable or necessary. Techniques for modifying nucleotide sequences using recombinant DNA methods are well known in the art.

The control sequence may be a suitable promoter sequence which is a nucleotide sequence recognized by the host cell for expression of that nucleotide sequence. The promoter sequence contains a transcriptional control sequence that mediates the expression of the peptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in selected host cells, including mutated promoters, truncated promoters, and hybrid promoters, and encodes extracellular or intracellular peptides that are homologous or heterologous to the host cell Can be obtained from a gene.

Examples of promoters that are particularly suitable for directing transcription of the nucleic acid constructs of the present invention in bacterial host cells include Escherichia coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis Levansukraase gene (sacB), Bacillus licheniformis alpha- Amylase gene (amyL), Bacillus stearothermophilus maltogen amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Baci-llus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lacta Promoters obtained from the horseshoe gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as tac promoters (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21- 25). Further promoters are “useful proteins from recombinant bacteria”: Scientific American (1980, 242: 74-94); And Sambrook et al., 1989 (homologous).

Useful promoters in yeast hosts include Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccha Obtained from the gene of -romyces cerevisiae 3-phosphoglycerate kinase Another useful yeast host cell promoter is described in Romanos et al., 1992, Yeast 8: 423-488.

The control sequence can also be a suitable transcription terminator sequence that is a sequence recognized by the host cell to terminate transcription. Terminator sequences are operably linked at the 3 'end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in the selected host cell can be used in the present invention.

Preferred terminators for yeast host cells are obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Terminators of other useful yeast host cells are described in Romanos et al., 1992 (homologous).

The control sequence can also be a suitable leader sequence that is an untranslated region of mRNA important for translation by the host cell. The leader sequence is operably linked at the 5 'end of the nucleotide sequence encoding the polypeptide. Any leader sequence that functions in the selected host cell can be used in the present invention.

Suitable leaders for yeast host cells are Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cere-visiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate It is obtained from the gene of dehydrogenase (ADH2 / GAP).

The control sequence can also be a polyadenylation sequence, a sequence operably linked to the 3 'end of the nucleotide sequence, which is a sequence that, when transcribed, adds a polyadenosine residue to the transcribed mRNA that is recognized by the host cell as a signal. Any polyadenylation sequence that functions in the selected host cell can be used in the present invention.

Polyadenylation sequences useful for yeast host cells are described in Guo and Sherman, 1995, Mole-cular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region, encoding an amino acid sequence linked to the amino terminus of the polypeptide and directing the encoded polypeptide to the cell's secretory pathway. The 5 'end of the coding sequence of the original nucleotide sequence contains a signal peptide coding region originally linked to the translation leading frame into a segment of the coding region encoding the secreted polypeptide. Alternatively, the 5 'end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. A foreign signal peptide coding region may be necessary if the coding sequence does not originally contain the signal peptide coding region. Alternatively, the foreign signal peptide coding region simply replaces the original signal peptide coding region to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used in the present invention.

Effective signal peptide coding regions for bacterial host cells include Bacillus NCIB 11837 maltogen amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stear-othermophilus neutral protease (nprT, nprT), nprS, And a signal peptide coding region obtained from the gene of Bacillus subtilis prsA.

Useful signal peptides for yeast host cells are obtained from genes of Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Another useful signal peptide coding region is described in Romanos et al., 1992 (homologous).

The invention also relates to recombinant expression vectors for the production of beta defensin comprising the nucleic acid constructs of the invention. The various nucleotides and control sequences described above may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of nucleotide sequences encoding peptides at those sites. Alternatively, the nucleotide sequence of the present invention can be expressed by inserting a nucleotide sequence or a nucleic acid construct comprising the sequence into a vector suitable for expression. In making an expression vector, a coding sequence is positioned in the vector such that the coding sequence is operably linked with a control sequence suitable for expression.

Recombinant expression vectors can conveniently carry out recombinant DNA processes and can be any vector (eg, plasmid or virus) that can result in expression of a nucleotide sequence. The choice of vector will typically depend on the compatibility of the vector and the host cell into which it will be introduced. The vector may be linear or closed cyclic prasmid.

The vector may be an autonomous replication vector, ie, a vector that exists as an extrachromosomal being, the replication of which is independent of chromosomal replication, for example a plasmid, extrachromosomal element, minichromosome, or artificial chromosome.

The vector may contain any means for ensuring self-replication. Or, when introduced into a host cell, the vector may be one that is integrated into the genome and replicates with the integrated chromosome (s). Moreover, two or more vectors or plasmids or transposons together containing a single vector or plasmid or total DNA to be introduced into the genome of the host cell can be used.

Vectors of the invention preferably contain one or more selectable markers that allow for easy selection of transformed cells. Selective markers are genes, the products of which provide biocides or virus resistance, resistance to heavy metals, protrophicity to nutritional supporters, and the like.

Examples of bacterial selectable markers are dal genes from Bacillus subtilis or Bacillus lichenif-ormis, or markers confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

The vectors of the invention preferably contain element (s) that allow for the stable integration of the vector into the genome of the host cell but permit the vector to autonomously replicate in the cell independently of the genome.

For integration into the host cell genome, the vector may depend on the nucleotide sequence encoding the peptide or any other element of the vector for stable integration of the vector into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences that direct integration into the host cell genome by homologous recombination. Additional nucleotide sequences enable the vector to integrate into the host cell genome at the correct place (s) in the chromosome (s). In order to increase the likelihood of incorporation in the right place, the integrating element is preferably a sufficient number of nucleotides, for example 100-1,500 base pairs, preferably 400-1,500 base pairs, and most preferably highly homologous to the corresponding target sequence. Should contain 800-1,500 base pairs, thereby enhancing the possibility of homologous recombination. The integration element can be any sequence that is homologous with the target sequence in the host cell genome. Moreover, the integrating element can be a non-coding or coding nucleotide sequence. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication that allows the vector to autonomously replicate in the host cell in question. Examples of bacterial origin of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC177, and pACYC-184, pUB110, pE194, pTA1060, and pAMf31 that allow replication in Bacillus. Examples of replication origin used in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The origin of replication may be with a mutation that makes its function temperature-sensitive in the host cell (see, eg, Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

One or more copies of the nucleotide sequences of the invention can be inserted into a host cell to increase the production of the gene product.

An increase in the number of copies of a nucleotide sequence can be obtained by incorporating at least one additional sequence copy into the host cell genome or by including an amplifiable selectable marker gene having a nucleotide sequence, in which case an amplified copy of the selectable marker gene Cells containing and additional copies of nucleotide sequences thereby can be selected by culturing the cells in the presence of a suitable selective agent.

The procedures used to construct the recombinant expression vectors of the invention by ligation of the elements described above are well known in the art (see, eg, Sambrook et al., 1989, homology).

The present invention relates to a recombinant host cell comprising the nucleic acid construct of the invention which is advantageously used in the recombinant production of beta defensin. A vector comprising the nucleotide sequence of the invention is introduced into a host cell, such that the vector is maintained as an chromosomal integration or self-replicating extrachromosomal vector as previously described.

The host cell may be a single cell microorganism, for example a prokaryote, or a non- single cell microorganism, for example a eukaryote.

Useful single cell cells are bacterial cells, for example gram positive bacteria, without limitation, Bacillus cells, for example Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Baci-llus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; Or Streptomyces cells, for example Streptomyces lividans or Streptomyces murinus, or Gram-negative bacteria, for example E. coli and Pseudomonas species.

Introduction of the vector into bacterial host cells can be accomplished by, for example, competent cells (e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, eg, Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (eg, Koehler and Thorne, 1987, Journal of Bacteriology 169 (5771-5278).

The host cell can be a eukaryote, for example a mammalian, insect, plant, or fungal cell.

In a preferred embodiment, the host cell is a fungal cell. As used herein, “fungus” is defined as that defined in the bacterium, basidiomycetes, staphylococcus, and junctures (Hawksworth et al., Ainsworth and Bisby's Dictionary of The Fungi (8th edition, 1995, CAB International, University Press, Cambridge, UK). As well as fungi (Hawksworth et al. (1995, homologous, cited on page 171)) and all mitosporic fungi (Hwksworth et al., Homologous).

In a more preferred embodiment, the fungal host cell is a yeast cell. As used herein, “yeast” includes vesicular spore forming yeast (capsulocytic yeast), vesicular spore forming yeast, and yeast (Blastomycetes) belonging to incomplete fungi. As the classification of yeast may change in the future, for the purposes of the present invention, yeast is known as Biology and Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, Soc. App. Bacteriol. Symposium Series No. 9). , 1980).

In a more preferred embodiment, the yeast host cell is a Candida, Hansenula, Kluyver-omyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells.

In the most preferred embodiment, the yeast host cell is a Saccharomyces cerevisiae cell.

Fungal cells can be transformed in a manner known in the art by processes involving protoplast formation, transformation of these protoplasts, and regeneration of the cell wall.

Yeast is Becker and Guarente, Abelson, J. N. and Simon, M. I. Editor, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; And Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

In addition, the present invention comprises the steps of (a) culturing a strain capable of producing the antibiotic peptide of the present invention; And (b) relates to a method for producing a peptide of the present invention, comprising recovering the peptide.

In addition, the present invention comprises the steps of (a) culturing a host cell under conditions conducive to the production of a peptide of the invention; And (b) relates to a method for producing an antibiotic peptide of the present invention, comprising recovering the peptide.

In the production method of the present invention, the cells are cultured in a nutrient medium suitable for producing peptides using methods known in the art. For example, cells may be shake flask culture, small or large scale fermentation (continuous, batch, fed-batch) or in laboratory or industrial fermentors, performed under conditions that allow the fermentation and / or separation of suitable media and peptides. Solid state fermentation). Cultivation takes place using procedures known in the art in suitable nutrient media including carbon and nitrogen sources and inorganic salts. Suitable media can be obtained from commercial suppliers or prepared according to published compositions (eg, catalogues of the American Type Culture Collection). If the peptide is secreted into nutrient medium, the peptide can be recovered directly from the medium. If the peptide is not secreted, it can be recovered from the cell lysate.

Peptides of the invention can be recovered by methods known in the art. For example, the peptide can be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

Peptides of the invention include, but are not limited to, chromatography (eg, ion exchange, affinity, hydrophobicity, chromatofocusing, and size exclusion), electrophoresis processes (eg, pre-isoelectric focusing), differential solubility (eg, For example, it can be purified by various procedures known in the art, including ammonium sulfate precipitation, SDS-PAGE, or extraction (eg, Protein Purification, JC Janson and Lars Ryden, Editor, VCH Publishers). , New York, 1989).

The invention also relates to compositions such as pharmaceutical compositions comprising the antimicrobial peptides of the invention.

The composition of the present invention comprises a peptide of the present invention.

The composition may further comprise other pharmaceutically active agents, for example additional fungicides, for example other antimicrobial polypeptides exhibiting the antimicrobial activity as defined above.

Fungicides can be antibiotics known in the art. Classes of antibiotics include penicillins such as penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, naphcillin, ampicillin and the like; Cephalosporins that are beta-lactamase inhibitors, for example penicillin in combination with cepacrol, cefazoline, cepuroxime, oxalactam and the like; Carbapenem; Monobactam; Aminoglycosides; tetracycline; Macrolides; Lincomycin; Polymyxin; Sulfoamides; Quinolone; Chloramphenicol; Metronidazole; Spectinomycin; trimethoprim; Vancomycin; And the like. Fungicides may also be anti-fungal agents and include polyenes such as amphotericin B, nistatin; 5-flucosine; And azoles such as myconazole, ketoconazole, itraconazole and fluconazole.

In one embodiment, the fungicide is a non-enzymatic chemical agent. In another embodiment, the fungicide is a non-polypeptide chemical agent.

Inventive antimicrobial peptides and fungicides of the compositions of the invention may be selected so that a synergistic antimicrobial effect is obtained. The antimicrobial peptides and fungicides of the composition are 50% w / w (preferably 25% w / w, more preferably 10% w / w, most preferably 5% w / w) and 0.5 ppm (preferably 0.1 ppm) Antimicrobial activity of at least 5% (preferably at least 10%) is higher than that obtained by adding the results of incubation separately using a fungicide and an antimicrobial peptide alone in an aqueous solution containing Can be selected to appear.

The composition of the present invention may comprise a suitable carrier material. The composition may comprise a suitable delivery excipient which, when used in medicine, can deliver the antimicrobial peptides of the invention to a desired location.

The composition may be prepared according to methods known in the art and may be in the form of a liquid or dry composition. For example, the peptide composition may be in the form of granules or microgranules. Peptides included in the composition can be stabilized according to methods known in the art.

Examples of preferred uses of the peptide composition of the present invention are given below. The dose of the peptide composition of the present invention and other conditions under which the composition is used may be determined based on methods known in the art.

The present invention also encompasses the various uses of antimicrobial peptides. Antimicrobial peptides are typically useful at any place contaminated by bacteria, fungi, yeast or algae. Typically, places are aqueous systems such as cooling water systems, laundry rinsing water, oil systems such as cutting oils, lubricants, oil fields and the like, and are places that need to kill microorganisms or inhibit their growth.

Other uses include preservation of food, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, limiting agents, deodorants, gargles, contact lens products, enzyme preparations, or food ingredients. .

Thus, the antimicrobial polypeptides of the present invention are antiseptics, for example in the treatment of acne, eye or mouth infections, skin infections; In limiting or deodorizing agents; In salts for foot baths; It is useful for cleaning and disinfecting contact lenses, hard surfaces, teeth (oral care), wounds and bruises.

The present invention also relates to the use of the antimicrobial peptide or composition of the present invention as a medicament. Furthermore, the antimicrobial peptides or compositions of the present invention can also be used in the manufacture of a medicament for controlling or killing fungal organisms or microorganisms, such as gram positive or negative bacteria.

Compositions and antimicrobial peptides of the invention can be used as therapeutic or prophylactic antimicrobial agents of veterinarians or humans. Thus, the compositions and antimicrobial peptides of the present invention can be used in the manufacture of a veterinary or human therapeutic or prophylactic for the treatment of microbial infections, such as bacterial or fungal infections, preferably gram positive or negative bacterial infections. In particular, microbial infections may be associated with, but not limited to, food poisoning and the like.

The composition of the present invention comprises an effective amount of the antimicrobial peptide of the present invention.

As used herein, the term "effective amount" means that the amount of the antimicrobial peptide of the present invention or fragment or variant thereof is sufficient to inhibit the growth of the microorganism in question.

Antimicrobial peptides of the invention can be prepared by in vitro synthesis using conventional methods known in the art. Various commercial synthesis apparatuses are available, for example an autosynthesizer by Applied Biosystems Inc., Beckman et al. Using synthetic groups, naturally occurring amino acids are substituted with unnatural amino acids, in particular D-isomers (or D-forms) such as D-alanine and D-isoleucine, diastereomers, side chains with different lengths and functional groups, and the like. Can be. The particular sequence and its mode of manufacture will be determined by convenience, economy, purity required, and the like.

Chemical linkages may be provided to various peptides or proteins, including functional groups that are convenient for binding, such as amide groups for the formation of amides or substituted amides, such as thiol groups for reducing amidation, thioethers or disulfide formation, and carboxyl groups for amide formation. Can be.

If desired, various groups can be introduced into the peptide during synthesis or expression that allow for linkage with other molecules or surfaces. Thus, cysteine can be used to make thioethers and histidines are linked to metal ion complexes, where carboxyl groups form amides or esters, and amino groups can be used to form amides.

In addition, peptides can be isolated and purified according to conventional methods for recombinant synthesis. Lysates can be prepared from expression hosts, and the lysates are purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In general, the composition used will comprise at least 20%, more typically at least about 75%, preferably at least about 95%, by weight, of the desired product, relative to the method of making the product and the contaminants associated with its purification, For professional purposes it will usually comprise at least about 99.5% by weight. Typically, percentages will be based on total protein.

The present invention also relates to a method of using a peptide having an antimicrobial activity in an animal feed, and to a feed composition and a feed additive comprising the antimicrobial peptide of the present invention.

The term "animal" includes all animals, including humans. Examples of animals are non-ruminants and ruminants such as pigs, cattle, sheep and horses.

In certain embodiments, the animal is a non-ruminant animal. Non-ruminant animals include, but are not limited to, single gastric animals such as pigs (including but not limited to piglets, growing pigs, and sows); Poultry such as turkeys and chickens (including but not limited to broiler chickens, egg laying chickens); Young calf; And fish (including but not limited to salmon).

The term "feed or feed composition" means any compound, preparation, mixture, or composition suitable for or intended for ingestion by an animal.

In use according to the invention, the antimicrobial peptides can be supplied to the animals before, after, or simultaneously with the diet.

In certain embodiments, the antimicrobial peptides in the form added to the feed or when included in the feed additive are well defined.

"Well defined" means that the antimicrobial peptide preparation is at least 50% pure as determined by size exclusion chromatography (see Example 12 of WO 01/58275). In another specific embodiment, the antimicrobial polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.

However, for use in animal feed, the antimicrobial peptide does not need to be pure, for example it may comprise other enzymes, in which case it may be termed an antimicrobial peptide preparation.

The antimicrobial peptide preparation of the present invention may be (a) added directly to the feed (or used directly in the processing of the vegetable protein), or (b) it is one or more added following the feed, such as feed additives or premixes. It may be used in the production of intermediate compositions (or used in the course of processing). The purity described above is considered to be the purity of the original antimicrobial peptide preparation, whether used according to any of (a) or (b) above.

Such antimicrobial peptide preparations can of course be mixed with other enzymes.

The antimicrobial peptides can be added to the feed in any form, either as relatively pure antibacterial peptides or in admixture with other ingredients added to the animal feed, ie in the form of animal feed additives, for example in the form of so-called premixes. Is added.

In a further aspect, the present invention relates to compositions for use in animal feed, such as animal feed, and animal feed additives such as premixes.

Apart from the antimicrobial peptides of the invention, the animal feed additives of the invention contain at least one fat soluble vitamin, and / or at least one water soluble vitamin, and / or at least one trace mineral, and / or at least one macro mineral. .

Furthermore, optional feed additive ingredients include colorants, fragrance compounds, stabilizers, and / or phytase EC 3.1.3.8 or 3.1.3.26; Xylanase EC 3.2.1.8; Galactanase EC 3.2.1.89; And / or at least one other enzyme selected from beta-glucanase EC 3.2.1.4.

Typically, fat-soluble and water-soluble vitamins, and trace minerals form part of the so-called premix, which is added to the feed, while large minerals are usually added separately to the feed.

In certain embodiments, the animal feed additive of the present invention is 0.01 to 10.0%; More preferably 0.05 to 5.0%; Or 0.2-1.0% (% means additive g per 100 g of feed), which is intended to be included (or prescribed for addition) in an animal diet or feed. This is especially true for premixes.

Examples of these components are listed below without limitation.

Examples of fat soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3. Examples of water soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and Pantothenates, for example Ca-D-pantothenate. Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt. Examples of large minerals are calcium, phosphorus and sodium.

Nutritional components of these components (exemplified by poultry and piglets / pigs) are described in Table A of WO 01/58275. Nutritional composition means that these ingredients must be provided in the diet at the concentrations indicated.

Alternatively, the animal feed additive of the present invention comprises at least one of the individual components specified in Table A of WO 01/58275. More specifically, this at least one individual component is included in the additive of the present invention in an amount capable of providing a concentration in the feed within the range shown in column 4, 5, or 6 of Table A.

The present invention also relates to an animal feed composition. Animal feed compositions or diets have a relatively high content of protein. The poultry and pig diets are characterized by the columns 2-3 of Table B of WO 01/58275. The diet of fish is characterized by the fourth column in Table B. Moreover, diets of such fishes usually have a crude fat content of 200-310 g / kg.

Animal feed compositions according to the invention have a crude protein content of 50-800 g / kg and further comprise at least one antimicrobial peptide claimed herein.

In certain embodiments, the animal feed composition of the present invention contains at least one vegetable protein or source thereof.

In another specific embodiment, the animal feed composition of the present invention comprises 0-80% corn; And / or 0-80% sugarcane; And / or 0-70% wheat; And / or 0-70% barley; And / or 0-30% oats; And / or 0-40% soy flour; And / or 0-10% fish flour; And / or 0-20% whey. Animal diets can be prepared, for example, as mash feed (non-pellet) or pellet feed. Typically, milled feed-raw materials are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species. The enzyme may be added as a solid or liquid enzyme preparation. For example, the solid enzyme preparation is typically added before or during the mixing step, and the liquid enzyme preparation is typically added after the pelleting step. The enzyme may also be mixed in feed additives or premixes.

The final enzyme concentration in the diet is in the range of 0.01-200 mg of enzyme protein per kg of diet, for example in the range of 5-30 mg of enzyme protein per kg of animal diet.

Antimicrobial peptides of the invention may be administered in one or more of the following amounts (dose ranges): 0.01-200; Or 0.01-100; Alternatively 0.05-100; or 0.05-50; Or 0.10-10. All of these ranges are in mg of antimicrobial peptide protein per kilogram of feed.

To determine the mg of antimicrobial peptide protein per kg of feed, the antimicrobial polypeptide is purified from the feed composition and the specific activity of the purified antimicrobial peptide is determined using the relevant assay (see the section on Antimicrobial Activity, Substrate, and Assay). .

The antimicrobial activity of the feed composition is also determined using the same assay, and based on these two measurements, the dose is calculated in mg antimicrobial peptide protein per kg feed.

The same principle applies to determining the antimicrobial peptide protein mg in feed additives. Of course, if the sample can utilize feed additives or the antimicrobial peptides used to make the feed, the specific activity is determined from this sample (no need to purify the antimicrobial peptides from the feed composition or additives).

As can be seen through the present invention it was possible to produce a large amount of beta defensin through the production method of the present invention, there is an effect that can be used in the pharmaceutical and feed industry through this by confirming the antibacterial activity through the mass produced defensin.

1 is a diagram showing the cDNA sequence confirmation and the amino acid sequence of the mature peptide through the NCBI gene bank database search for beta defensin 2.
Figure 2 is a beta defensin mature peptide nucleotide sequence amplification and construct vector.
Figure 3 is a bacterial count after the culture by transfection of vectors containing put in dependency pig antimicrobial peptide of 2 to 293T cell, and then creating a lysate collected by cell inoculation with Salmonella Enteritidis bacteria were cultured at 4 ℃ and 37 ℃ after 24 hours Figure measured.
4 is transfected with a vector containing the pig antimicrobial peptide defensin of FIG. Figure 24 shows the bacterial counts after incubation at.
Figure 5 is a schematic diagram showing the introduction process, to secure the PCR amplification product using a construct prepared to extract each mature peptide fragment as a template.
Figure 6 is a construct expression vector prepared for extracting each mature peptide fragment.
Figure 7 is a picture of the disk diffusion to perform the PBD1, 2, 3 antibiotic stimulation response in bacteria. / L3262, L1 on the right represents pGAL10-PBD1 / Y2805 ㅿ gal1, L2 represents pGAL10-PBD2 / Y2805 ㅿ gal1, L3 indicates pGAL10-PBD3 / Y2805 ㅿ gal1, y: Yeast / DW (-) control and Am Ampicillin (+) control.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example 1 Construction of Pig Beta Defensin Gene Expression System

1) pig beta defensin information

Pig beta defensin information is shown in Table 1.

① Pig Beta Defensin 2 (PBD2)

In order to utilize yeast as an expression, the beta defensin was identified by cDNA sequence and amino acid sequence of mature peptide through NCBI gene bank database search. In particular, by comparing the beta defensin gene structure of pigs and humans, the mature peptide sequence required for construct construction was reconfirmed (Fig. 1).

As can be seen in Figure 1, beta defensin 2 in pigs and humans has a similar structure. Although the mature peptide composition was somewhat different, disulfide bonds expected to play a role as antimicrobial function were found to be identical.

2) Expression vector construction for swine beta defensin expression: Tissue nonspecific expression vector using beta-actin promoter (FIG. 2)

pCAGGS vector with beta-actin promoter (promoters expressed in whole tissues) is expressed in mammalian cells, and cell lysate obtained by transfection of 293T cells to antibacterial activity This vector was used for this purpose. This vector is a vector obtained by newly processing the promoter portion purchased from addgene (USA).

3) Amplification of pig beta defensin mature peptide sequence and construction of construct

PCR primers for each mature peptide amplification were designed and amplified. Primer design sequences required for amplification are as follows (Table 2). The primer of the present invention was used by the manufacturer of Bioneer Korea.

Porcine lung cDNA was prepared by the following method.

Total RNA was extracted using TRIzol (Invitrogen, USA) from 0.3 g of pig lung. 3 μg of total RNA, oligo- (dT) ₁₅ Primer (Promega, USA), 10 mM dNTP Mix (TAKARA, Japan) were mixed, denatured at 65 ° C. for 5 minutes, cooled on ice for 5 minutes, and then denatured RNA. The mixture was mixed with 5X First-strand buffer, 0.1M DTT, Superscript III ^TM reverse transcriptase (Invitrogen, USA), and obtained cDNA after 4 minutes at 50 ° C 53 minutes, 72 ° C 15 minutes, and 4 ° C.

For PCR amplification, the reaction solution contained 10X reaction buffer containing cDNA and 10 mM MgCl ₂ in pig lung, 10 pmol of each primer, 25 mM dNTP, and 1.25U of Pyrobest Taq polymerase (TAKARA, JAPAN). It was. The amplification using each primer was heated for 3 minutes at 94 ° C to induce denaturation, followed by 35 steps of 30 seconds denaturation at 94 ° C, 30 seconds of primer annealing at 58 ° C or 55 ° C, and 3 steps of 1 minute at 72 ° C. Extension was performed at 72 ° C. for 4 minutes. PCR amplification products were purified using Gel Extraction kit (Qiagen, USA) by cutting specific DNA bands from 365nm UV lamp after gel electrophoresis for 40 minutes under 1X TAE running buffer, 100V on 1.5% agarose gel. Purified DNA was treated for 2 hours at 37 ° C. using 20 U Xba I, Xho I (TAKARA, JAPAN) restriction enzyme, and the vector was also digested with Xba I, Xho I restriction enzyme for introduction into pCAGGS vector (FIG. 2). DNA digested with restriction enzyme and pCAGGS vector were ligated at 16 ° C. for 16 hours using T4 DNA ligase 350 U (TAKARA, JAPAN). The ligated DNA was transformed into E. coli DH10B cells (Invitrogen, USA) using electroporation. After 1 hour of incubation in a 37 ° C. shaker incubator, the cells were inoculated into LB plates to which empicillin (50 μg / ml) was added and incubated for about 16 hours in a 37 ° C. incubator. After 16 hours DNA was extracted from a single colony formed on the plate (GeneAll, Korea). The extracted DNA was sequenced in both directions on an ABI 3730 DNA analyzer (Applied Bissystems, USA) with the ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, USA). Sequencing results were analyzed in NCBI.

4) Effect of swine defensin on Salmonella and Escherichia coli

Salmonella Enteritidis (SE) Antimicrobial Effect Experiment: After transfection of pig antimicrobial peptide defensin into 293T cells (Invitrogen, USA), the cells were harvested to make lysate and antibacterial against Salmonella Enteritidis and E.coli 0157: H7 (1000CFU). The effect was found. After inoculating the prepared lysate and incubated at 4 ℃ and 37 ℃ to determine the number of bacteria after 24 hours.

24 hours later 4 ℃ a lysate to be a result of Salmonella Enteritidis PBD 1, 2 more control measures the bacteria cultured in 3 both showed that reducing the number of bacteria Salmonella Enteritidis. The number of bacteria incubated at 37 ° C was also lower than in the control. Porcine antibacterial peptide defensin shows an antimicrobial effect (Fig. 3).

After 24 hours, the number of Ecoli O157: H7 bacteria was decreased in the lysate cultured at 4 ° C. The bacterial counts of those cultured at 37 ° C. were also lower than controls (FIG. 4).

Example 2: Antimicrobial Activity Assay of Porcine Beta Defensin Expression Product Using Yeast

To test the antimicrobial efficacy of lysate through expression in the yeast cytoplasm of pig beta defensin, yeast strain expression vectors were constructed, introduced into yeast strains, and the final product was cultivated by culturing the introduced yeast strains and extracting lysate. The function was confirmed.

1) Amplification of pig beta defensin mature peptide sequence and introduction of the vector in the expression strain

Design PCR primers for each mature peptide amplification and amplify them. Each primer design sequence required for amplification is as follows (Table 3).

PCR amplification products were obtained by using the constructed construct as a template to extract each mature peptide fragment, the introduction process and the contents of the Park Hyun vector are as follows (Figs. 5 and 6). Cloning to a new expression vector using the above Primer was confirmed, and introduced into the yeast strain.

2) Transformation in Yeast Strains

PGAL10-PBD1 was transformed into S. cerevisiae Y2805 ㅿ gal1 and L3262 strains (F.D. Life Science, Korea). 5 ~ 10ng of pGAL10-PBD1 and 100㎍ denatured sheared salmon sperm DNA, PEG / LiAC solution (40% PEG-3350, 1X TE, 1X LiAC) was added and incubated for 30 minutes in a shaker at 30 ℃. After 30 minutes DMSO is added and heat shock is applied at 42 ° C. for 7 minutes. After centrifugation at 2500 rpm for about 10 seconds, the supernatant is discarded and the pellet is dissolved in 1X TE. Dissolved pellets were inoculated in YNBCAD (0.67% YNB, 2% glucose, 0.5% casamino acids, 2% Agar) plate and incubated for 3-4 days in a 30 ℃ incubator.

3) disk diffusion test

Disc diffusion was performed to view PBD1 antibiotic stimulation in bacteria.

YPDG (1% Yeast extract, 2% peptone, 1% glucose, 1% galactose) cultures were inoculated with a single colony of S. cerevisiae Y2805 ㅿ gal1, L3262 transformed with each pGAL10-PBD1, and in a 30 ° C shaking incubator. Incubated for about 16-24 hours. Centrifuge the culture at 1500 g for 5 minutes, dissolve in Y-per Yeast protein extraction reagent (Thermo, USA) and centrifuge again. The supernatant was removed, and the pellet was dissolved with Y-per Yeast protein extraction reagent to confirm the turbidity (OD600> 50-100). Then, the same amount of glass beads (acid-washed) was added.

<110> Konkuk University Industrial Cooperation Corp. <120> Method for producing antimicrobial peptide and use of the same <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> mPBD-2F <400> 1 tctagagcca gaggatggac cactac 26 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PBD-2R <400> 2 ctcgaggtgg cttctggctc a 21 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> N-PBD2 <400> 3 accctcgacg aattcatggg tcttggccag aggtccgacc ac 42 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> C-PBD2 <400> 4 atacgctccg tcgactcagc ggatgcagca cttggc 36 <210> 5 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 5 Gly Leu Gly Gln Arg Ser Asp His Tyr Ile Cys Ala Lys Lys Gly Gly   1 5 10 15 Thr Cys Asn Phe Ser Pro Cys Pro Leu Phe Asn Arg Ile Glu Gly Thr              20 25 30 Cys Tyr Ser Gly Lys Ala Lys Cys Cys Ile Arg          35 40 <210> 6 <211> 349 <212> DNA <213> Artificial Sequence <220> <223> PBD2 <400> 6 cctctgagag cctctccttc cctgggcatc cggccaccac ctgccgccgc catgagggcc 60 ctctgcttgc tgctgctgac tgtctgcctc ctctcttccc agctggctgc aggtattaac 120 ctgcttacgg gtcttggcca gaggtccgac cactacatat gtgccaagaa aggggggacc 180 tgcaacttct ccccctgccc gctcttcaac aggattgaag ggacctgtta cagtggcaag 240 gccaagtgct gcatccgctg accctgagcc agaagccaca gcagagggga cacacaggcc 300 aagtgaagta tttgcaactg ttgttttaat aaaagaagtt tttttttga 349

Claims (10)

a) performing PCR using a primer for amplifying beta-diffsin using cDNA derived from porcine tissue as a template;
b) restriction enzyme treatment of said PCR product;
c) ligation of the restriction enzyme treated PCR product into the expression vector of FIG. 2; And
d) transforming the expression vector into a host cell.
In Vitro Expression of Porcine Beta Defensin. The method of claim 1, wherein the primer has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 2. In vitro expression method of porcine beta defensin. The method of claim 1, wherein the beta-defensin has an amino acid sequence of SEQ ID NO: 5 in vitro expression method of pig beta defensin. a) performing PCR using a primer for amplifying beta-diffsin using cDNA derived from porcine tissue as a template;
b) restriction enzyme treatment of said PCR product;
c) constructing the expression vector of FIG. 6 by ligating the restriction product treated PCR product to an expression vector; And
d) transforming the expression vector into a host cell.
In Vitro Expression of Porcine Beta Defensin. The method of claim 4, wherein the primer has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3 and 4. The method of claim 4 wherein the beta-defensin has an amino acid sequence of SEQ ID NO: 5 in vitro expression method of porcine beta defensin. According to claim 4, wherein said host cell is an in vitro expression method of porcine beta defensin, characterized in that the yeast. An antimicrobial composition against pathogenic microorganisms comprising beta-defensin prepared by the expression method of any one of claims 1 to 7. The antimicrobial composition of claim 8, wherein the pathogenic microorganism is Gram-positive or Gram-negative bacteria. The antimicrobial composition of claim 9, wherein the Gram-positive bacteria is Bacillus cereus or Staphylococcus aureus, and the Gram-positive bacteria is Escherichia coli or Salmonella enteritidis.

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