KR20050028129A - Bovine lactoferricin expressed in plants - Google Patents

Abstract

Bovine lactoferricin expressed in plants is provided, which bovine lactoferricin is easily mass-produced, and has improved antimicrobial activity against various microorganisms, so that the bovine lactoferricin can be useful in medicines, feed and cosmetics. A polynucleotide encoding the bovine lactoferricin has the nucleotide sequence set forth in SEQ ID NO:3. A plant expression vector of bovine lactoferricin comprises (1) the polynucleotide of SEQ ID NO:3; (ii) a promoter operately linked to the polynucleotide of (i) and forms an RNA molecule in plant cells; and (iii) 3-non-translated region resulting in polyadenylation of the RNA molecule in plant cells. A method for producing the recombinant bovine lactoferricin in plants comprises the steps of: (a) inserting the plant expression vector of bovine lactoferricin into the plant cells; (b) selecting the transformed plant cells; (c) regenerating a plant from the transformed plant cells; and (d) producing the recombinant bovine lactoferricin in the regenerated plant.

Description

Bovine Lactoferricin Expressed in Plants

The present invention relates to lactoferricin expressed in plants, and more particularly, to novel polynucleotides encoding lactoferricin, a vector comprising the same, a plant into which the vector is introduced, and a method for producing lactoferricin using the plant. It is about.

Mammalian milk (milk), tears, saliva, nasal secretions, and genital mucosa contain antibacterial antibiotics that kill externally invading bacteria. Called lactoferrin, the substance consists of about 700 amino acids. When pepsin is applied to lactoferrin under acidic conditions, an antimicrobial peptide is isolated. This peptide is called lactoferricin. Lactoferricin is composed of 66 amino acids and is an important component of the defense system against foreign antigens. It is a peptide substance that is about 500 times stronger in antimicrobial activity than lactoferrin.

Lactoferrin is a type of transferrin, a protein that binds to iron. The first lactoferrin was identified in cow milk, which was subsequently isolated from milk of several mammals, including rats, goats, rabbits, dogs, pigs, and humans.

Lactoferricin exists in an iron-saturated state with an iron-free state, which deprives iron ions necessary for survival of microorganisms in the state where iron is not bonded, thereby preventing the growth of microorganisms. It is suppressed and killed. In addition, synergistic effect of bacteriostatic action is shown in cooperation with lysozyme or immunoglobulin, and it has been reported to cause bactericidal effect through direct interaction with cell membrane components of microorganisms. However, it has been reported that it does not cause antimicrobial activity against bacteria useful for the human body, such as Lactobacillus and bifidus. It is also known to work on viruses that are fatal to humans such as HIV or hepatitis C virus.

Thus, lactoferricin has the advantage of being excellent in terms of side effects because it is a bactericidal effect against bacteria and is inherently in vivo when compared to conventional antibiotics. Therefore, although it has a very advantageous advantage in the development of food, cosmetics or pharmaceuticals, the actual commercialization is still a small stage. It is a small peptide with lactoferricin, and because of its high ratio of lysine and arginine with a positive charge in the composition of amino acids, and because lactoferricin itself has antibacterial activity, it is difficult to express lactoferricin directly in microorganisms. This is because there are few cases of successful expression.

The present inventors have made diligent research efforts to solve the long-standing needs of the art described above, and have successfully introduced novel polynucleotides encoding bovine-derived lactoferricin into plants, thereby obtaining lactoferricin showing antimicrobial activity. By confirming that the present invention can be completed.

It is therefore an object of the present invention to provide novel polynucleotides encoding bovine-derived lactoferricin.

Another object of the present invention is to provide a plant expression vector of recombinant bovine-derived lactoferricin.

It is still another object of the present invention to provide a method for preparing a transient transfected plant that expresses recombinant bovine-derived lactoferricin temporarily.

Another object of the present invention is to provide a transgenic plant which transiently expresses recombinant bovine-derived lactoferricin.

Another object of the present invention is to provide a method for preparing recombinant bovine-derived lactoferricin.

Another object of the present invention is to provide a method for producing a transgenic plant stably expressing recombinant bovine-derived lactoferricin.

Another object of the present invention is to provide a transgenic plant stably expressing recombinant bovine-derived lactoferricin.

Another object of the present invention is to provide a recombinant bovine-derived lactoferricin produced by the method of the present invention.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a polynucleotide encoding a lactoferricin comprising a nucleotide sequence described in SEQ ID NO: 1, 3 or 5 sequence.

Sequence Listing The first sequence is for human-derived lactoferricin, the third sequence is for bovine-derived lactoferricin, and the fifth sequence is for pig-derived lactoferricin.

The novel polynucleotides of the present invention encode lactoferricin and have a modified nucleotide sequence to optimize expression in plants without amino acid substitutions.

The design of the polynucleotides of the invention is carried out by the following criteria: (i) to make the GC content at least 50%, (ii) to have the plant's preferred codons, and (i) to remove intron-like sequences. will be. Such plant-compatible sequences can increase the translation rate of genes (Kusnadi et al ., Biotechnol. Prog. 14: 149-155 (1998)). In addition, the intron-like sequence in the foreign gene to be introduced is removed in order to rule out the possibility that the arrangement of the transgene is recognized as an intron in the host plant, resulting in cleavage in the plant nucleus and a failure to produce the desired protein.

The novel polynucleotides of the present invention include the nucleotide sequences described in SEQ ID NO 1, 3 and 5 sequences as well as some sequences thereof. That is, when the amino acid sequence encoded by the above partial sequence exhibits antimicrobial activity, which is a representative activity of lactoferricin, it is included in the polynucleotide of the present invention.

In addition, the novel polynucleotides of the present invention are to be interpreted to include nucleotide sequences which show substantial identity to the nucleotide sequences described above, as well as the nucleotide sequences set forth in SEQ ID NO: 1, 3 and 5 sequences. The substantial identity is that at least 90% of the phases when the nucleotide sequence of the present invention and any other sequence are arranged to correspond as closely as possible and the sequence is analyzed using algorithms commonly used in the art. By nucleotide sequence, which shows homology, more preferably 95% homology, most preferably 98% homology.

According to another aspect of the present invention, the present invention provides a kit comprising (i) a nucleotide sequence set forth in SEQ ID NO: 1, 3, or 5; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be any one commonly used in the art for gene introduction of a plant, for example, the Coliflora Mosaic Virus (CaMV) 35S promoter , Nopaline synthase (nos) promoter, pigwarm mosaic virus 35S promoter, sugacran basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carboxylase small sub Photoinducible promoters of ssRUBISCO, rice cytosol triosphosphate isomerase (TPI) promoters, adenine phosphoribosyltransferase (APRT) promoters of Arabidopsis and octopine synthase promoters.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al., Nucleic Acids Research , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tuberculosis, of the protease inhibitor I or II gene of tomato or potato 3 'terminal portion and CaMV 35S terminator.

Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention is a selection marker antibiotics (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) resistant gene (e.g., neomycin phospho-transferase dehydratase (nptⅡ), high thereof Mycin phosphotransferase ( hpt ), and the like).

According to the present invention, the production of plants into which the gene encoding lactoferricin is introduced is largely achieved in two ways: transient transfected plants and transgenic plants.

As used herein, the term "transient transfected plant" means that the introduced gene is not transferred to the next generation as the plant into which the foreign gene has been introduced. In transiently transfected plants, foreign genes are generally present separately from the chromosome of the host cell. In contrast, the term “transgenic plant” refers to a plant having a foreign gene that is transmitted to the next generation. In transgenic plants, foreign genes are generally integrated into the chromosomes of the stromal cells to become the genetic repertoire of the host cell, and reliably delivered to the next generation.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into the plant cell of the plant; And (b) provides a method for producing a transient transfected plant to temporarily express recombinant lactoferricin comprising the step of confirming whether the gene has been introduced into the plant cells.

According to another aspect of the present invention, the present invention provides a transient transfected plant that transiently expresses recombinant lactoferricin prepared by the above method.

According to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into plant cells; (b) confirming whether the gene has been introduced into the plant cell; And (c) obtaining recombinant human lactoferricin from a plant comprising the plant cell into which the gene is introduced.

In the method of the present invention, transient transfection of plant cells can be carried out according to conventional methods known in the art (Rainer Fisher et al., Biotechnol. Appl. Biochem. , 30: 113-116 (1999) ). Transient gene expression in plants is faster than in transformants with stable expression, and the result of protein expression can be confirmed as soon as possible. Therefore, whether the target protein obtained in plants prior to large-scale production using stable transgenic plants is functioning normally. Suitable for checking

Therefore, the present inventors expressed, isolated and purified lactoferricin using a transient gene expression method in plants for efficient mass production of lactoferricin having antimicrobial activity.

Transient gene expression methods are suitable approaches to test the functionality and stable expression of target genes before manufacturing plant transformants for mass production, which has the advantage of saving time and money. In particular, plant transformants produced by stable transformation show a chromosomal positional effect that is affected by the position at which the foreign gene is inserted. Transient transfection can rule out this effect. It is well suited for the efficient expression and isolation of foreign genes.

In transient transfection, there are three types of methods for delivering foreign genes to plant cells: particle bombardment, which coats and introduces naked DNA onto particles (Christou, P., Trends Plant Sci). 1: 423-431 (1996)), vacuum infiltration (vacuum infiltration) Agrobacterium-fill trafficking Orientation method (agroinfiltration containing the expression vector is introduced into plant tissue by Agrobacterium in the like) (Kapila et al, plant Sci. ., 122: 101-108 (1996)), and a modified plant virus virus vector method using a vector (viral vectors) (Scholthof, H. et al, Annu Rev. Phytopathol 34:... 299-323 (1996) ).

The transformation efficiencies of these three methods vary, so that in the case of DNA introduction by particle guns, genes are generally introduced into only a small number of cells and function only when DNA reaches the cell nucleus for transcription. This method can be used to test the stability of recombinant proteins prior to stable transformation, but is not suitable for mass expression of recombinant proteins.

Agroinfiltration allows genes to be introduced into more cells than the particle gun, whereby T-DNA containing the target gene is actively introduced into the nucleus with the help of several bacterial proteins. In this method, the introduced T-DNA is not integrated into the host cell's chromosome and continuously expressed, but is present in the nucleus independently and expresses the transient expression of the gene of interest. It is suitable for obtaining protein quickly.

In addition, the method using a viral vector introduces a foreign gene into the genome of a viral plant pathogene to be controlled by a strong promoter, and when the recombinant viral vector including the foreign gene is infected with a plant, the foreign gene introduced is During plant virus replication, they are amplified with high efficiency and then expressed to produce foreign proteins. However, when using a viral vector, the size of the protein to be produced is limited to less than 30 kDa, so the size of the gene to be introduced is difficult to use.

Therefore, in the present invention, the agroin fill method is most preferable. The agroin filtration method is generally carried out with leaves.

When the present invention is practiced in agrofiltration, more preferably, the Agrobacterium tumefaciens -binary vector system is used.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') Agrobacterium tumefaciens containing a vector having a sequence of Infiltrating tissue: a nucleotide sequence set forth in SEQ ID NO: 1, 3, or 5 sequence; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts in plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') confirming the expression of the protein in the infiltrated leaves.

In one specific embodiment described above, suitable leaves include any tissue derived from the germinated seed, preferably the leaves produced before the peduncle rises, most preferably the leaves are too young or not mature. The younger the leaves, the higher the expression level, but the tissues are more susceptible to yellowing or dying. Seed germination is carried out under suitable dark / light conditions using a suitable medium.

Introduction of genes into plant cells is carried out with Agrobacterium trimers containing Ti plasmids (Depicker, A. et al., In Genetic Engineering of Plants, Plenum Press, New York (1983)). More preferably, binary vector systems such as pBin19, pRD400, pRD320 and pHS737 are used (An, G. et al., “Binary vectors” In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986); RK Jain, et al., Biochem. Soc. Trans. 28: 958-961 (2000).

Gene introduction into the plant cells in the leaves by Agrobacterium tufasion includes methods known in the art. Most preferably, the gene introduction process includes infiltration of a culture of Agrobacterium tumeration into the tissues of the leaves.

The infiltration process may be performed by a vacuum method or a syringe method. One specific embodiment when using a vacuum is as follows. The plant leaves are placed in a culture of Agrobacterium trimmers and vacuum is applied for a short time. Quickly release the vacuum, disinfect the leaves with sterile water, and place them on a damp piece of paper with the top of the leaves facing down. The leaves are then stored at about 22 ° C. under light conditions of about 16 hours. Then, after about 2-3 hours, the expressed target protein is identified.

On the other hand, a specific embodiment when using a syringe is as follows. The back of the plant leaves is infused with a syringe of Agrobacterium turmeration using a syringe, the plant is incubated for about 3-5 days, and then the expressed target protein is identified.

Agrobacterium tuberation cultures preferably include acetosyringone, to aid in the penetration of Agrobacterium into plants.

Plants transgenic according to the present invention are confirmed the expression of the protein of interest by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transgenic plants to identify foreign genes present in plant cells. Alternatively, Northern or Southern blotting can be performed to confirm gene introduction (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). On the other hand, if the vector used to introduce the gene has a β-glucuronidase gene, a part of the leaf tissue into which the gene is introduced is selected from X-gluc (5-Bromo-4-Chloro-3-Indole-β-D- By visually observing the color development by immersion in a substrate solution such as Glucuronic acid) it can be easily confirmed whether the gene introduced (Jefferson, RA, Plant Mol. Biol. Rep. , 5: 387 (1987)).

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

Lactoferricin can be obtained from the tissue of the transgenic plant of the present invention, that is, the transiently transfected plant (eg, leaves), and the extract obtained from the tissue can be obtained through a conventional purification process. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

In a specific embodiment of the present invention, when there is 6 x His at the N-terminus of the recombinant lactoferricin of the present invention, a Ni-NTA His-binding resin column is used to quickly and easily separate and remove the desired recombinant lactoferricin. It can be purified.

According to the method of the present invention, not only can the recombinant lactoferricin be produced in a short period of time at low cost by using a transient transfected plant, but the produced lactoferricin exhibits the same antimicrobial activity as natural-occurring lactoferricin.

Another approach for preparing plants in which genes encoding lactoferricin has been introduced is to use transgenic plants that stably express lactoferricin.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; And (c) provides a method for producing a transformed plant stably expressing the recombinant lactoferricin comprising the step of regenerating the plant from the transformed plant cells to obtain a transformed plant.

According to another aspect of the present invention, the present invention provides a transgenic plant stably expressing the recombinant lactoferricin produced by the method of the present invention described above.

According to another aspect of the invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining recombinant lactoferricin from the transgenic plant.

According to another aspect of the present invention, the present invention provides a recombinant lactoferricin produced by the above method.

In the method of the present invention, the transformation of plant cells can be carried out according to a conventional method known in the art, which is electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982) And Saul, M. et al., Plant Molecular Biology Manual , Vol. A1, Kluwer Academic Publishers, Dordrecht (1988)), particle gun method (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568- 9572 (1990) and Christou, P., Trends Plant Sci. 1: 423-431 (1996)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011). Of these, Agrobacterium-mediated transformation is most preferred. Agrobacterium-mediated transformation is generally carried out with leaf discs and other tissues (cotyledon and hypocotyls). Agrobacterium-mediated transformation is most efficient in dicotyledonous plants.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selective resistance are grown and divided in the cultures described above. Exemplary labels include, but are not limited to, glycophosphate resistance genes and neomycin phosphotransferase (nptII) systems.

Methods of developing or regenerating plants from plant protoplasts or various implants are well known in the art (Bhojwani, SS et al., Plant Tissue Culture: Theory and Practice , Elsevier Science, New York, (1983); and Lindsey, K., Ed., Plant Tissue Culture Manual , Kluwer Academic Publishers, Dordrecht, The Netherlands, (1991)). The transformed root shoots formed are imbedded in a suitable plant growth medium. Development or regeneration of plants comprising foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

On the other hand, the present inventors have made extensive research efforts to develop novel transgenic plants (eg, tobacco, melons, cucumbers, watermelons and rapeseeds), and as a result, have established the most efficient method for the transformation of specific plants. Such a method is disclosed in PCT / KR02 / 01461, PCT / KR02 / 01462 and PCT / KR02 / 01463, which patent documents are incorporated herein by reference.

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens -binary vector system.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') an Agrobacterium tumer embedded in a genome DNA of a plant cell and having a vector having the sequence Infecting the plant's plant with Agrobacterium tumefaciens : (i) the nucleotide sequence encoding the lactoferricin described in SEQ ID NO: 1, 3 or 5; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; (Iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') re-differentiating the infected implant in a regeneration medium to obtain a redifferentiated shoot; And (c ') culturing the re-differentiated shoots in a rooting medium to obtain a transformed plant.

In one specific embodiment described above, suitable implants for transformation include any tissue derived from the germinated seed, preferably cotyledon and hypocotyl, most preferably cotyledon. Seed germination is carried out under suitable dark / light conditions using a suitable medium. Transformation of plant cells is carried out with Agrobacterium trimers comprising Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983) ).

More preferably, binary vector systems such as pBin19, pRD400 and pRD320 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986)). Binary vectors suitable for include (i) a promoter operating in a plant, (ii) a structural gene operably linked to said promoter, and (iii) a polyadenylation signal sequence Optionally, said vector comprises a reporter molecule (eg : Additionally carries genes encoding luciferase and glucuronidase) Examples of promoters used in binary vectors include the CaMV 35S promoter, 1 promoter, 2 promoter and nopaline synthase (nos) promoter. It is not limited to this.

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process comprises coculture by impregnating cotyledon in a culture of Agrobacterium tumerification. As a result, Agrobacterium trimmers are infected into plants.

Explants transformed with Agrobacterium trimerization are re-differentiated in regeneration medium, which successfully leads to regeneration of shoots. Finally, transgenic plants are produced by the rooting of shoots that have been regenerated in the rooting medium.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genome of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

The lactoferricin expressed in the transformant can be obtained from tissues derived from various organs (e.g., stems, leaves, roots, fruits, seeds, etc.) of the transformed plant, and the extract obtained from the tissues is subjected to conventional purification. You can get it. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

According to the method of the present invention, not only can lactoferricin be produced in large quantities at low cost, but the produced lactoferricin exhibits the same antimicrobial activity as natural-occurring lactoferricin.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1 Synthesis of Novel Genes of Lactoferricin Peptides

24 (human-derived lactoferricin), 26 (bovine-derived lactoferricin) or 24 (pork-derived lactoferricin) amino acids (see SEQ ID NO: 2), identical to the native lactoferricin gene Sequence, the fourth sequence and the sixth sequence), but unlike the nucleotide sequence encoding the existing lactoferricin, the optimal nucleotide sequence designed by the plant was designed. The design criteria for the new gene is first, to make the GC content above 50%, to have the plant's preferred codons, and to remove intron-like sequences. The nucleotide sequences thus designed were chemically synthesized at the Plant Biotechnology Institute (hereinafter referred to as "PBI") and the National Research Center (SK, Canada). DNAs encoding the three recombinant lactoferricin peptides derived from synthesized human, bovine and swine are described in SEQ ID NO: 1, 3 and 5, respectively.

Example 2: Preparation of a plant expression vector of the lactoferricin gene

Synthetic genes encoding the recombinant lactoferricin peptide are described in SEQ ID NOs: 1, 3 and 5, and synthesized to have a recognition site of Xba I at the 5'-end and Bam HI at the 3'-end. .

PHS737 (PBI, National Research Center, Canada), a plant expression binary vector, was digested with restriction enzymes Xba I and Bam HI enzymes, digested, and isolated and purified. The synthesized gene and the cleaved pHS737 vector were mixed, and then, the ligation buffer (KOSCO, Korea) and T4 ligase (KOSCO, Korea) were added to the mixture and reacted at 16 ° C. for at least 1 hour. The reaction was then transformed into competent cells E. coli strain DH5α (Promega, USA) treated with CaCl ₂ and antibiotic resistant strains were selected in LB medium containing the antibiotic kanamycin (100 mg / ml).

Plasmid DNA was isolated from the selected clones using an alkaline method, and human lactoferricin was purified by forward primer 5′-AAT CTA GAA ATG GCT ACT AT-3 ′ and reverse primer 5′-AAG GAT CCC CGA GCT TGA GA-3 ′ , Bovine lactoferricin is forward primer 5'-AAT CTA GAA ATG GCT ACT AT-3 'and reverse primer 5'-AAG GAT CCC CGA GCT TGA GA-3', porcine lactoferricin is forward primer 5'-AAT CTA GAA ATG Polymerase chain reaction (PCR) was performed using GCT ACT AT-3 'and reverse primer 5'-AAG GAT CCC CGA GCT TGA GA-3', respectively. The enzyme used in the PCR amplification was Taq polymerase (Solgent, Korea), and the temperature conditions were set as follows: predenature for 2 minutes at 96 ° C, denature for 1 minute at 94 ° C, and connect for 1 minute at 55 ° C. The reaction was repeated 35 times at 72 ° C. for 2 minutes, and further extended at 72 ° C. for 10 minutes.

The amplification product was then analyzed on a 1.0% agarose gel to confirm that the desired insert sequence was in the plasmid.

Example 3: Incorporation of Lactoferricin into Plants

Example 3-1 Preparation of Agrobacterium tumefaciens GV3101 Culture

Conjugation of the recombinant pHS737 / hLF vector cloned with each human-derived lactoferricin, the recombinant pHS737 / bLF cloned with bovine-derived lactoferricin, or the recombinant pHS737 / pLF vector cloned with pig-derived lactoferricin, prepared in Example 2 (conjugation) Was introduced into Agrobacterium turmerics GV3101 (Mp90), respectively ( Plant-cell-rep. , 15 (11): 799-803 (1996)). Escherichia coli S17-1 and Agrobacterium Tumperfections GV3101 (Mp90) with a recombinant pHS737 vector containing lactoferricin were incubated up to logarithmic phase in liquid LB medium, respectively. Each of these cells were mixed together 1: 1 in an Eppendorf tube and then centrifuged for 30 seconds to concentrate the cells, followed by stationary culture at 1-2C for 1-2 days. The Eppendorf tube containing the cells over time was gently shaken to float the concentrated cells, and then plated in LB solid medium to which 50 mg / L kanamycin and 30 mg / L gentamicin were added. Colonies were selected after incubation for one day (Alt-Morbe et al ., J. Bacteriol. 178: 4248-4257 (1996)). The strains into which the vectors were introduced were named Agrobacterium turmerics GV3101-pHS737 / hLF, GV3101-pHS737 / bLF and GV3101-pHS737 / pLF.

Agrobacterium tumersions GV3101 containing pHS737 cloned with the lactoferricin gene was inoculated in liquid LB medium and incubated at 28 ° C. for 2 days to be used as a seed. The fully cultured spawn was added to 1/50 of the final culture volume and antibiotic (50 mg / L kanamycin) in the liquid LB medium and incubated for about 18 hours until the absorbance was reached at 600 nm. The culture solution was centrifuged at 3500 rpm for 15 minutes at room temperature to remove the supernatant, and then the cells were suspended in a 10 mM MgCl ₂ solution (containing 0.2 ppm of BA and 150 μM of acetosyringone) and allowed to acclimate at room temperature for at least 3 hours. It was used for infection of plants.

Example 3-2 Preparation and Agroinfiltration of Plant Cells

Seeds of the first sterilized tobacco ( Nicotina bentamiana ) were sown and incubated for at least 3 weeks and then purified in a greenhouse. Agrobacterium turmerics GV3101 (Mp90) containing a pHS737 vector with a lactoferricin gene on the leaves of the purified plant was prepared by the method described in Example 3-1. After inserting into (Cross), it was injected until it spreads evenly on the back of the leaf. If the fungus is injected into the whole leaf, the leaf is heavy and the plant itself may fall down or the leaf disease may break.

Plants infused with Agrobacterium return to their original condition after 10 minutes to 1 hour. Plants injected with Agrobacterium were harvested within 5 days after 3 days of culture and analyzed using the method of Example 4.

Example 4 Label Factor Analysis for Confirming Transgene Expression in Transgenic Plants

Confirmation of gene expression of the plant secured in Example 3 was performed as follows: To confirm the expression of beta-glucuronidase (β-glucuronidase, GUS) reporter gene, leaves were harvested. 200 μl of the following GUS assay solution (X-gluc (5-Bromo-4-Chloro-3-Indole-β-D-Glucuronic Acid), 1 mg / ml) was placed in a container with leaves and a vacuum was used. Keep vacuum until it is completely wet. After that, the appearance of color was visually observed to confirm the introduction and expression of the gene.

When the gene of the leaf to which the gene is introduced is expressed, the beta-glucuronidase, a reporter gene, is produced, and the enzyme beta-glucuronidase is produced. When the X-gluc solution is added, the beta-glucuronidase is added. As the X-gluc is broken down, pigments are produced, causing the cells to appear blue. In the present invention, the leaf into which the gene is introduced, the reporter gene beta-glucuronidase is expressed to indicate the blue color to confirm the introduction and expression of the gene.

Example 5: Isolation and Purification of Recombinant Lactoferricin Peptide in Transgenic Plants

In a mortar containing an appropriate amount of liquid nitrogen, 1 g of the leaves of the plant in which the genes were introduced were cut out and crushed. Finely ground was added 2 ml of protease inhibitor-containing extract buffer (250 mM sucrose, 1 M Hepes, 1 mM MgCl ₂ , 1 mM DTT) per 0.5 g of plant weight. The extract was centrifuged at 12,000 rpm and 4 ° C. for at least 30 minutes, then the supernatant was removed and transferred to a new tube. This lysed cell solution was added to a Probond column (Quiagen GmbH, Germany) containing nickel resin, allowing the recombinant lactoferricin peptide in the cell solution to adsorb well to the nickel resin while gently shaking at room temperature for 10 minutes.

The resin to which the recombinant lactoferricin peptide was adsorbed was washed twice with the same amount of pH 7.8 adsorption buffer solution (20 mM sodium phosphate and 500 mM sodium chloride) to remove plant cell derived proteins adsorbed on the resin. An equal amount of pH 6.0 wash buffer solution (20 mM sodium phosphate and 500 mM sodium chloride) was added to the washed resin and gently shaken at room temperature to wash the resin again. The same amount of pH 4.0 elution buffer solution (20 mM sodium) was added. Phosphate and 500 mM sodium chloride) were added to elute the recombinant lactoferricin protein.

Recombinant lactoferricin peptides expressed in transgenic plants have six histidine residues at the amino terminus and are readily isolated by binding to a cationically nickel resin. Through this process, only recombinant lactoferricin protein from which all plant cell proteins were removed was purified. The eluted protein was concentrated using Centricon (Amicon Co., U.S.A.).

Recombinant lactoferricin peptides were electrophoresed on SDS-PAGE gels and stained with Coomassie Blue, and recombinant lactoferricin peptides, which were not detected in wild-type tobacco, were detected in cell eluates, especially when eluted with a nickel resin column. It was confirmed that the pure recombinant lactoferricin peptide with all cellular proteins removed was detected.

Example 6: Antimicrobial Assay of Recombinant Lactoferricin Peptide

In order to confirm the antimicrobial activity of recombinant lactoferricin protein isolated and purified from the leaves of plants expressing genes introduced by agroinfiltration, the antimicrobial activity was measured against various strains such as Gram-positive bacteria, Gram-negative bacteria and fungi, yeasts. It was. E. coli O-157 KCTC 1682, Pseudomonas aeruginosa KCTC 1637, Salmonella choleaesuis supsp.choleraesuis KCTC 2932, Bacillus subtilis ( Bacillus) subtilis KCTC 102), Streptococcus mutans KCTC 3065, Staphyloccocus aureus KCTC 1621, Listeria monocytogenes KCTC 3710, Saccharomyces cerevisiae 7 ), Candida albicans KCTC 7270, Mycosporum canis KCTC 6591, and the like.

Colonies of each strain were selected and cultured for 18 hours in LB medium, and then the harvested cells were diluted to 600 with an absorbance of 0.6 at 600 nm in 1 mM MgCl ₂ buffer. This was inoculated with 5 ml of 0.6% agar solution to 10 ⁴ cfu, and then evenly spread on LB solid medium.

     For the antimicrobial assay, a disc containing ampicillin at concentrations of 10 μg / ml and 1 μg / ml was used as a positive control. Each lactoferricin was used as a 50 μg purified liquid extracted from genetically introduced tobacco, and a protein extract of wild-type tobacco was used as a negative control. The bacterial culture solid medium on which each disk is placed is placed in an incubator suitable for the growth temperature of each strain and incubated for 8 hours, and then visually examined for colony formation, clear zone and diffusion zone formation. Observation of the result is shown in Table 1. Positive when the inhibitory ring was 10 mm or more.

Strains Human lactoferricin Solactoferricin Porcine Lactoferricin Gram-negative bacteria Escherichia coli ++ + ++ Klebsiella pneumoniae + + + Pseudomonas aeruginosa + + + Salmonella typhimurium + + + Gram-positive bacteria Bacillus subtilis + + ++ Streptococcus pneumoniae + + + Staphyloccocus aureus + + + Listeria monocytogenes + + + Fungus Saccharomyces serevisiae + + + Aspergillus flavus + + +

In Table 1, "+++", "++" and "+" are cases where the inhibitory ring is 25 mm or more, 20-25 mm and less than 20 mm, respectively, and "-" is no growth inhibition.

As can be seen in Table 1, human, bovine and pig-derived lactoferricin of the present invention did not show a significant difference in antimicrobial activity, and showed antimicrobial activity against various strains such as Gram-positive bacteria, Gram-negative bacteria and fungi, yeasts.

On the other hand, Figure 1a shows that the human-derived lactoferricin of the present invention inhibits the growth of Pseudomonas aeruginosa and Bacillus subtilis to show an inhibitory ring. Figure 1b shows that the bovine-derived lactoferricin of the present invention inhibits the growth of Pseudomonas aeruginosa and Bacillus subtilis, indicating an inhibitory ring. In addition, Figure 1c shows that the pig-derived lactoferricin of the present invention inhibits the growth of Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis to show inhibitory ring.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described in detail above, the present invention provides a novel polynucleotide encoding lactoferricin and a plant expression vector of recombinant lactoferricin comprising the same. The present invention also provides a transient transfected plant that transiently expresses recombinant lactoferricin and a transformed plant that stably expresses recombinant lactoferricin. On the other hand, the present invention provides a method for producing recombinant lactoferricin. According to the present invention, not only can lactoferricin be produced in large quantities at low cost, but the produced lactoferricin shows excellent antimicrobial activity against various microorganisms, and thus can be widely used in medicine, feed, and cosmetics.

1A-1C are photographs showing the antimicrobial activity of human-derived lactoferricin, bovine-derived lactoferricin and pig-derived lactoferricin, respectively, of the present invention.

<110> NEXGENE BIOTECHNOLOGIES, INC. <120> Bovine Lactoferricin Expressed in Plants <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> the nucleotide sequence of human lactoferricin <220> <221> CDS (222) (1) .. (72) <400> 1 atg aca aag tgc ttc caa tgg cag agg aat atg agg aag gtg cgt ggc 48 Met Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly 1 5 10 15 cct cct gtc agc tgc atc aag agg 72 Pro Pro Val Ser Cys Ile Lys Arg 20 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <400> 2 Met Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly 1 5 10 15 Pro Pro Val Ser Cys Ile Lys Arg 20 <210> 3 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> the nucleotide sequence of bovine lactoferricin <220> <221> CDS (222) (1) .. (78) <400> 3 atg ttc aag tgc cga cga tgg caa tgg agg atg aag aag act ctg ggt gct 48 Met Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala 1 5 10 15 cca tct atc act tgt gtg agg agg gcc ttc 78 Pro Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25 <210> 4 <211> 26 <212> PRT <213> Artificial Sequence <400> 4 Met Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala 1 5 10 15 Pro Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25 <210> 5 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> the nucleotide sequence of porcine lactoferricin <220> <221> CDS (222) (1) .. (72) <400> 5 atg tcc aag tgc cgc cag tgg caa tct aag atc aga aga aga acg aat ccc 48 Met Ser Lys Cys Arg Gln Trp Gln Ser Lys Ile Arg Arg Thr Asn Pro 1 5 10 15 atc ttc tgc atc agg agg gcg tcg 72 Ile Phe Cys Ile Arg Arg Ala Ser 20 <210> 6 <211> 24 <212> PRT <213> Artificial Sequence <400> 6 Met Ser Lys Cys Arg Gln Trp Gln Ser Lys Ile Arg Arg Thr Asn Pro 1 5 10 15 Ile Phe Cys Ile Arg Arg Ala Ser 20

Claims (9)

A polynucleotide encoding bovine-derived lactoferricin comprising the nucleotide sequence set forth in SEQ ID NO: 3 sequence. (Iii) the polynucleotide of claim 1; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule. A method for preparing a transient transfected plant that expresses recombinant bovine-derived lactoferricin temporarily comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell of a plant; And (b) confirming whether the gene has been introduced into the plant cell. A transient transfected plant transiently expressing recombinant bovine-derived lactoferricin prepared by the method of claim 3. A method for preparing a recombinant bovine-derived lactoferricin comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) confirming whether the gene has been introduced into the plant cell; And (c) obtaining recombinant lactoferricin from a plant comprising plant cells into which the gene has been introduced. A method of producing a transgenic plant stably expressing recombinant bovine-derived lactoferricin comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant. A transgenic plant stably expressing recombinant bovine-derived lactoferricin prepared by the method of claim 6. A method for preparing a recombinant bovine-derived lactoferricin comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining recombinant lactoferricin from the transgenic plant. Recombinant bovine-derived lactoferricin prepared by the method of claim 5 or 8.