KR20020080731A - Lactoferricin gene and transformant expressing lactoferricin - Google Patents

Abstract

PURPOSE: A lactoferricin gene and a transformant for the expression of lactoferricin are provided, therefore the lactoferricin having antimicrobial, antiviral and anti-inflammatory activity can be easily mass-produced. CONSTITUTION: The lactoferricin genes derived from a deer, a dog and a rabbit have the nucleotide sequence of SEQ ID NOs: 1, 3 and 5, respectively. They have the amino acid sequence of SEQ ID NOs: 2, 4 and 6, respectively. Recombinant vectors pKSSI, pKSDO and pKSRA are constructed by inserting the lactoferricin derived from a deer, a dog and a rabbit into an expression vector pPIC9, respectively. Transformants Pichia pastoris SLCS(KCTC 0956BP), Pichia pastoris SLCD(KCTC 0954BP) and Pichia pastoris SLCR(KCTC 0955BP) are produced by transforming Pichia pastoris GS115 with the recombinant vectors pKSSI, pKSDO and pKSRA, respectively. The method for producing lactoferricin comprises the steps of: culturing the transformants Pichia pastoris SLCS(KCTC 0956BP), Pichia pastoris SLCD(KCTC 0954BP) or Pichia pastoris SLCR(KCTC 0955BP) in a medium containing 4% glycerol for 24 hours; preinducing the cultured medium with 0.5% ethanol for 4 hours; and introducing 1 to 4% of 99% ethanol into the cultured medium and culturing it at 26 to 30 deg. C for 72 hours.

Description

Lactoferricin gene and transformant expressing lactoferricin using the same {Lactoferricin gene and transformant expressing lactoferricin}

The present invention relates to a lactoferricin gene and a transformant for expressing lactoferricin containing the same. More specifically, the present invention isolates the lactoferricin gene encoding lactoferricin protein, which has been reported antimicrobial activity, anticancer activity and synergism with antibiotics, from deer, dogs, rabbits and is expressed in expression vectors and peachia. The present invention relates to a transformant prepared by introducing into a Pastoris yeast strain.

Recently, the misuse and persistence of antibiotics in the livestock, food and medicine fields, that is, the antibiotic residue of livestock foods and the resulting resistance is very serious. Therefore, there is a growing demand for natural antibiotics used to secure the stability and productivity of livestock products and to treat diseases caused by bacterial infection.

Peptides having antipathogenic and antimicrobial activity are already known in the art, and soluble peptides such as mammalian defensin, secropin, thionine, melittin and insect defensins are known and inhibit fungal growth. Hydrolysable enzymes such as chitinase and β-1,3-glucanase are also known as another class of peptides having antipathogenic activity.

Lactoferrin (LF), which is secreted from mammals, is also called lactotransferrin, and together with serotransferrin, ovotransferrin, siderophilin (natural iron-binding glycoprotein) It is a type of iron-binding protein classified as a family. Lactoferrin is a non-immunoglobulin that is particularly found in colostrum in mammals and is also found in saliva, tears, mucus and endocrine glands [Masson, P. L. and J. F. Heremans, Biochem. Biochem. Biophy Acta. 257-314 (1971). Lactoferrin protects against harmful microorganisms [Arnold, RR, RMCole., JRMc Ghee., Science 197, 263-265 (1977)], and promotes intestinal iron absorption in infants [Nemet.K., I.Simonovits, Haematologia ( 1985)], regulation of myelopoiesis [Broxemeyer. HE, M. De Soysa., P. Ralph, Blood (1980)], decreased mastitis incidence [Tsul.S., hirata.Y., Mukai.F. , Ohtagaki. S., Dairy Sci. 152 125-128 (1990)], promoting protein synthesis in liver [Burrin, D.C., Wang, H., Heath, J., Dudley, M.A., Pediatr. Res. 40, 72-76 (1996)], growth promoting effect of lymphocytes [Ambruso.DR, RBJohnston, Invenst 67, 357 (1981)], generation of hydroxyl by neutrophils [Anderson.WL, TB Tomasi, Atch. Biochem. Biophys. 182, 705 (1977)], reduction of lysozyme regeneration [Bullen. J.J., J.A.Amstrong, Immunology. 36, 781 (1979) and macrophage, granulocytes, neutrophils, leukocytes, and antiviral effects [Masnori I., Akito] N., Kazuo S. Torahiko T., Atsushi N., Katsuki T. Hisahiko S., Kunitada S., Masaki S., Nobuyuki K., Virus Research 66, 51-63 (2000)]. These lactoferrins are Gram positive, negative bacteria as well as fungi [Botner, C.A., R.D. Miller., R.R.Arnold, Immun. 51, 373-377 (1986)], because lactoferrin, like many other transferrins, inhibits microbial growth by depriving and cheating iron ions that microorganisms need. to be. In addition, it has been reported that lactoferrin is involved in killing by releasing lipopolysaccharide in the outer membrane of Gram-negative bacteria and destroying the stability of the membrane and enhancing cell permeability [Richard T. Ellison III, The effects of lactoferrin on gram-negative bacteria. Lactoferrin: Structure and function. 71-90 (1994). Lactoferrin also synergizes with other proteins such as lysozyme and immunoglobulin A, which is the result of the aggregation of lactoferrin into the cell wall of bacteria modified by lysozyme [Ellison, RT, TJGiehl., FWLactorce, Immunol, 56, 2774 (1988).

Lactoferrin, which was first isolated from milk by Groves, entered the 1990s by molecular biological research, revealing the lactoferrin cDNA sequences and structures of various animals, including humans. Lactoferrin consists of a single polypeptide divided into two lobes (C and N), the two lobes connected by a short α-helix. Each lobe has two domains (N lobe N1, N2, C lobe C1, C2) and an ion binding site in the cleft between domains. Tomita discovered that peptides obtained by treating lactoferrin with pepsin, a protease, showed stronger antimicrobial activity than the original lactoferrin and named the peptide lactoferricin (LFc), which is called human lactoferricin. Nucleotide sequences of bovine lactoferricin were identified [Tomita.M., Bellamy.W., Takase.M., Yamauchi.K., Wakabayashi.H., Kawase.KJ, Dairy Sci. 74, 4137-4142 (1992). Recently, lactoferricin was produced in the stomach of lactoferrin-induced adults, and it was confirmed through clinical experiments that lactoferricin activity concentration was sufficient to have antimicrobial effect in the stomach of infants digested with milk [Kuwata.H. , Yip. TT., Tomita. M., Htchens. TW., Biochem. Biophys. Acta. 1429, 129-141 (1998).

Lactoferricin digested by pepsin lies on the surface of the N1 domain of lactoferrin, and it has been reported that bovine lactoferricin has a much greater antimicrobial activity than human and bovine lactoferrin by 300-500 times [Richard. T., Ellison. III, The effects of lactoferrin on gram-negative bacteria. Lactoferrin: Structure and function. 71-90 (1994). In addition, lactoferricin has shown excellent efficacy in anticancer effect experiments in rats, and also synergistic with antibiotics such as penicillin G, vancomycin, gentamicin, and erythromycin. Have been reported [Lars H. Vorlan, Svein aA. Osbak, Torunn Perstolen., J. Infect Dis 31, 173-177 (1999).

There are two hypotheses that lactoferricin has a higher antimicrobial activity than lactoferrin, which contains lactoferrin but is located in the N1 domain, which does not contain an ion binding site. First, in lactoferrin, lactoferricin is partially exposed to the surface, which confers microbial membrane inhibition on lactoferrin. However, because lactoferrin is larger in size than lactoferricin, it interferes with the interaction between the lactoferricin moiety and the microbial membrane. Second, lactoferricin inside lactoferrin has a helix-turn-sheet motif, whereas lactoferricin isolated from lactoferrin is converted to antiparallel β-sheet and has an amphipathic structure that is more suitable for microbial binding [David J. Schibi , Hans J. Vogel., Structural studies of lactoferricin B and its antimicrobial active peptide fragments. Lactoferrin: Structure, Function and application, 27-35 (2000)].

Pichia pastoris used as a gene expression host in the present invention belongs to the genus of methylotrophic yeast together with the genus Hansenula , Candida , Torulopsis . In the early 1970s, methylotrophic yeast was used to produce single cell protein (SCP) using methanol as a carbon and energy source. In the 1980s, the fast methanol metabolism of Peachia pastoris produced more cell mass, and about 60% of the cell mass was expressed as protein, so Peachia pastoris was used in the stabilized fermentation industry [Wegner, GH]. , W. Harder., Methylotropic yeast. In: Van Verseveld, HW, JA Duineeds. Microbial growth on C1 compounds: proceeding of the 5th international symposium. Boston: Martinus Nijhoff. 131-138 (1986)]. Methanol is a very efficient system for producing recombinant genetic material because of its ability to precisely regulate expression by methanol, use AOX1, a promoter with high expression rate, and high cell culture. come. Methanol oxidation occurs in membrane-enclosed organells called peroxisomes, which can multiply during methanol metabolism and make up about 80% of the cytoplasm. In addition, the microbial eukaryote not only has advantages for eukaryotic expression systems such as protein processing, protein folding and posttranslational modification, but also more than other eukaryotic expression systems such as baculovirus or animal cells. It can be used quickly, easily and cheaply. In addition, there is an advantage that can be expressed in a large amount (10-100 times or more) of a high quality heterologous protein (heterologous protein). This action is made possible by inserting a gene encoding a heterologous protein under the AOX1 promoter required when using methanol. The use of methanol as a carbon source under the AOX1 promoter causes methanol to act as an inducer, resulting in active transcription of the gene encoding the heterologous protein sandwiched therebetween, resulting in the desired external gene product ( A large amount of foreign gene products can be obtained. In addition, Pchia Pastoris has very similar characteristics to Saccharomyces cerevisae, a yeast that is widely used in industrial and genetic engineering fields. Therefore, it is very easy to use molecular biological, genetic and biochemical technologies. Therefore, many techniques related to transformation, gene disruption and gene replacement developed in Saccharomyces cerevisia can be applied to Peachia pastoris as it is. In addition, since Pchia pastoris releases a very small amount of native product into the media, most of the protein released in the actual media becomes a heterologous protein, which is the first step in protein purification. This may be an advantage of reducing the first step [Barr, KA, SAHopkins, K. Sreekrishna. Pharm. Eng. 12: 48-51 (1992). For this reason, in the present invention, a histidine-required strain Pchia pastoris GS115 strain was selected as a host for expression of lactoferricin and used in the transformation process.

Medicines, research on industrial mass production until spite of lactoferricin God in demand with a variety of features, such as oil industry, food additives, cosmetic additives and feed additives, and still can not see hardly find recent Aspergillus (Aspergillus) in Only a few studies are being conducted on the expression of lactoferrin through the human body [PPWard, JYLo, GSMay, Biotechnology 10: 784-789 (1992)]. Therefore, the present inventors newly isolated the lactoferricin coding gene showing an antimicrobial activity almost similar to antibiotics from the spleen and liver tissues of deer, dog and rabbit whose nucleotide sequence has not yet been identified. . In addition, by applying genetic engineering method to be the basis of industrial application using microorganism, subcloning the lactoferricin gene of the deer, dog, and rabbit identified in the sequence into the expression vector, and introducing the recombinant vector into yeast by lactoferricin gene. As a result, the transgenic strain expressing the lactoferricin of the deer, dog, rabbit was prepared. In other words, it does not receive a big impact on lactoferricin god with superior antimicrobial activity against microorganisms unicellular eucaryote widely used in the fermentation industry yeast master p Chiapas pastoris (Pichia pastoris) the selection of the host cell by deer, dog, rabbit-derived Expression of lactoferricin was attempted.

Accordingly, it is an object of the present invention to provide DNA and amino acid sequences encoding lactoferricin of deer, dogs and rabbits. Another object of the present invention is a recombinant vector constructed by inserting a gene encoding lactoferricin of deer, dog, and rabbit into an expression vector pPIC9, and transforming lactoferricin produced by introducing the same into a Pchia pastoris GS115 yeast strain. To provide a sieve. It is another object of the present invention to provide a lactoferricin protein expressed from the lactoferricin expression pichia pastoris transformant.

The object of the present invention is to isolate and amplify the lactoferricin coding gene by RT-PCR using primers of total RNA isolated from spleen and liver tissues of deer, dogs, rabbits, and liver tissues, and primers KS1E and KS-Eco. After analyzing DNA and amino acid sequences, the recombinant vector was prepared by digesting the gene and the pPIC9 subcloning vector derived from E. coli Top10F 'by Xho I and EcoR I restriction enzymes and ligating with T4 DNA ligase. Introduced by electroporation into the histidine-required strain Pychia pastoris GS115 yeast strain lacking histidine dehydrogenase activity to produce a transformant that expresses lactoferricin, and then expressed the recombinant lactoferricin expressed therefrom. This was accomplished by measuring the antimicrobial activity of various strains of proteins and confirming their expression patterns.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a cleavage map of the expression vector pPIC9 of the present invention.

Figure 2 shows the electrophoresis of isolated total RNA of deer, dogs, rabbits.

Figure 3 is a result showing the electrophoresis pattern of 2% agarose gel electrophoresis of lactoferricin cDNA obtained by performing RT-PCR using the total RNA of the deer, dog, rabbit KS1E and KS-Eco primer.

4 shows the results of PCR amplification of pKSSI, pKSDO, and pKSRA using KS1E and KS-Eco primers.

Figure 5 shows the results of PCR re-amplification of pKSSI, pKSDO, pKSRA using 3 'AOX and KS1E primers.

Fig. 6 is a schematic diagram and a cleavage map showing a process of constructing a recombinant vector pKSSI in which a deer lactoferricin gene is inserted into an expression vector pPIC9 vector.

7 is a schematic diagram and a cleavage map showing a process of constructing a recombinant vector pKSDO in which dog lactoferricin gene is inserted into an expression vector pPIC9 vector.

Fig. 8 is a schematic diagram and a cleavage map showing a process for constructing a recombinant vector pKSRA in which a rabbit lactoferricin gene is inserted into an expression vector pPIC9 vector.

9 is a DNA and amino acid sequence of the lactoferricin of the deer, dog, rabbit of the present invention.

10A and 10B compare the homology of the lactoferricin amino acid sequences of the deer, dog, and rabbit of the present invention with lactoferricin amino acid sequences of cows, pigs, goats, horses, camels, humans, foxes, etc., which have been identified. Alignment (A) and phylogenetic tree (B) results shown.

11A and 11B show the secondary structure map (A) and helical wheel (B) of the deer lactoferricin of the present invention by the Chou-Fasman algorithm. Where * represents a cationic peptide.

12A and 12B show the secondary structure map (A) and helical wheel (B) of the dog lactoferricin of the present invention by the Chou-Fasman algorithm. Where * represents a cationic peptide.

Figure 13 shows the secondary structure map (A) and helical wheel (B) of the rabbit lactoferricin of the present invention by the Chou-Fasman algorithm. Where * represents a cationic peptide.

14 is a screening result of Mut ⁺ and Mut ^s transformants of the present invention SLCS.

15 shows screening results of Mut ⁺ and Mut ^s transformants of the SLCD of the present invention.

16 is a screening result of Mut ⁺ and Mut ^s transformants of the present invention SLCR.

17A and 17B show the results of SDS-PAGE (A) and Western blot (B) analysis of culture concentrates of the present invention SLCS, SLCD, and SLCR.

In the present invention, the liquid nitrogen is put into the liver tissues of the deer and the dog's spleen and earthenware, and then ground. After mixing RLT, EtOH, RPE buffer, and DEPC treated water, the total RNA is separated by centrifugation and confirmed by UV transmission. step; Amplifying lactoferricin cDNA of deer, dogs, and rabbits by performing RT-PCR using KS1E and KS-Eco primers as templates for the total RNA; The RT-PCR product was electrophoresed and extracted with a 220bp sized band, which is estimated to be a lactoferricin gene, PCR was again performed as a template, and a 220bp sized band was extracted to obtain insert DNA of deer, dog, and rabbit lactoferricin. Making; Obtaining the subcloning vector pPIC9 from E. coli Top10F ′; Ligation of the deer, dog, and rabbit lactoferricin gene (cDNA of LFc) and pPIC9 by restriction enzymes Xho I and EcoR I followed by mixing with T4 DNA ligase and 10 × buffer; The ligated mixture was added to E. coli DH5α competent cells cultured in LB medium supplemented with ampicillin, transformed by heat shock treatment, and plasmids were extracted from the cultured colonies to extract 3'AOX, KS1E, and KS-. PCR amplification and electrophoresis with Eco primers to confirm ligation; Re-amplification using 3'AOX and KS1E primers to confirm ligation in the forward direction, and naming the recombinant vectors into which the lactoferricin genes of deer, dog, and rabbit were inserted were named pKSSI, pKSDO, and pKSRA, respectively; Analyzing the DNA and amino acid sequences of the recombinant vectors pKSSI, pKSDO, and pKSRA of the present invention and comparing homology with known lactoferricin sequences of bovine, human, goat, horse, pig, and camel foxes; Analyzing the amino acid sequence of the deer, dog, rabbit lactoferricin using an algorithm of DNASIS and analyzing the secondary structure of the lactoferricin protein; Cutting the recombinant vectors pKSSI, pKSDO, and pKSRA into Sal I to make a linear mixture, and then mixing them with Pichia pastoris GS115 and transforming them by electroporation; Culturing the transformant in histidine-deficient medium to select strains His ⁺ SLCS, SLCD, SLCR having lactoferricin genes of deer, dog, and rabbit; PCR using 5'AOX1 and 3'AOX1 primers as a template of the genomic DNA of the His ⁺ SLCS, SLCD, SLCR transformants was performed, and the methanol metabolism ability of expressing a large amount of lactoferricin in His ⁺ transformants was excellent. Selecting Mut ⁺ ; Culturing the transformant His ⁺ SLCS, SLCD, SLCR strains of the present invention to obtain a lactoferricin culture medium and measuring antimicrobial activity against various strains such as gram (+), gram (-) bacteria and fungi; SDS-PAGE and Western blot analysis is performed to confirm the expression patterns and expression levels of lactoferricin of deer, dog and rabbit expressed from the transformants SLCS, SLCD, and SLCR of the present invention.

In this step, modifying enzymes such as enzyme, T4 DNA ligase and Klenow fragment were used by Takara (Japan), Boehringer Mannheim (Germany) and Posco (Korea). Medium was LB broth (5% yeast extract, 10% tryptone, 5% NaCl, Difco), YPD (1% yeast extract, 2% peptone, 2% dextrose, Difco), MD (1.34% YNB, 4 × 10 ^-5 % Biotin, 1% Dextrose), MM (1.34% YNB, 4 × 10 ^-5 % Biotin, 0.5% Methanol), BMGY (1% Yeast Extract, 2% Peptone, 1.34% YNB, 1% Glycerol 100 mM potassium phosphate, pH 6.0, 4 × 10 ⁻⁵ % biotin), BMEY (1% yeast extract, 2% peptone, 1.34% YNB, 0.5% ethanol 100 mM potassium phosphate, pH 6.0, 4 × 10 ⁻⁵ % biotin) . Other reagents were purchased from Sigma (USA), Duchefa (Netherlands), and Difco (USA).

In this step, the deer for identifying the lactoferricin (LFc) sequence was purchased from Myeongcheon Deer Farm in Chungbuk-dong, Manchurian silka deer , and the dog ( Canis lupus domesticus species) was obtained from Chungnam National University Animal Hospital, Rabbit ( Lion head). Species) were purchased from Chungnam National University Animal Hospital and used respectively.

In this step, a pPIC9 (Invitrogen) vector derived from E. coli Top10F 'was used as a cloning expression vector for subcloning the lactoferricin gene. The characteristics of the pPIC9 vector are shown in Table 1, and a cleavage map is shown in FIG.

Characteristics of the Expression Vector pPIC9 of the Invention designation Description Feature 5'AOX1 With AOX1 promoter Allow methanol-inducible high level expression in Pichia.Targets plasmid integration to the AOX1 locus. S Coding N-terminal Protein Secretion Signals Isolates the desired protein of interest MCS Multi Cloning Site Allow insertion of target gene TT Transcription termination Permits efficient transcription termination HIS4 Site coding for histidiol dehydo genase (2.4kb) Provides a selectable marker to isolate recombinant strains 3'AOX1 Sequences from AOX1 gene that are further 3 'to the TT sequences Targets plasmid integration at the AOX1. Amp Ampicillin resistence gene Selectable. ColE1 E.coli replication origin E.coli origin of replication Let it replicate

E. coli DH5α (Invitrogen) was used as a strain for amplification of the plasmid, and Pichia pastoris GS115 (Invitrogen) was used as a host cell for expression of lactoferricin. The strains and plasmies of the present invention are summarized in Table 2.

Strains and Plasmids of the Invention Strain / plasmid Description Source E. coli Top10F ＇(pPIC9) (F＇lacIqTn10Tet ^R ) mcrA (nrr-hsdRMS-mcrBC) Φ80lacZΔM15 lacX74deoRrecAIaraD139 Δ (ara-leu) 7697galUgalKrpsL endA1nupG), containing pPIC9 Invitrogen DH5α supE44 △ lacU169 (ψ80lacZ △ M1) hsdR17 recA1endA1 gyrA96 thi-1 relA1 Invitrogen Yeast ( Pichia pastoris ) GS115 his4 ^-, mut ^+, containing AOXgene Invitrogen SLCS HIS4 ⁺ , mut ⁺ , GS115 with pKSSI The present invention SLCD HIS4 ⁺ , mut ⁺ , GS115 with pKSDO The present invention SLCR HIS4 ⁺ , mut ⁺ , GS115 with pKSRA The present invention Plasmid pPIC9 Amp ^r , HIS4 ⁺ , AOX1 promoter pKSSI Amp ^r , HIS4 ⁺ , AOX1 promoter, containing 220bp deer lactoferricin The present invention pKSDO Amp ^r , HIS4 ⁺ , AOX1 promoter, containing 220bp dog lactoferricin The present invention pKSRA Amp ^r , HIS4 ⁺ , AOX1 promoter, containing 220bp rabbit lactoferricin The present invention

In this step, E. coli was incubated with LB broth or plate at 37 ° C., and 50 μg / ml of ampicillin was added if necessary. Peachia pastoris was cultured using YPD plate, transformants were screened using MD and MM plate. For the expression of the transformants SLCS, SLCD, SLCR of the present invention was shaken at 30 ℃ using BMGY, BMEY medium.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Isolation of Lactoferricin Gene from Animal Tissues

First step. Isolation and Identification of Total RNA from Deer, Dog and Rabbit

Take 100mg of fresh deer, dog, rabbit liver and spleen and store it in liquid nitrogen, place it in a cooled mortar and put it in liquid nitrogen to crush it. Total RNA RNeasy kit (QIAGEN Co., Cat.No. 74104) USA) was used to isolate total RNA. Into the 600 μl of RLT buffer (10 μl β-mercaptoethanol / 1 mL RLT) 30 mg of the ground tissue was homogenized and centrifuged (12,000 rpm, RT, 3 minutes). EtOH (70%) was added to the supernatant, mixed, and then transferred to a 2 mL collection tube equipped with a micro-membrane tube and centrifuged (12,000 rpm, 4 ° C., 15 seconds). The column was washed with 700 μl of RW1 buffer, followed by 500 μl of RPE buffer (RPE: EtOH = 1: 4), followed by centrifugation (12,000 rpm, 4 ° C., 15 seconds). The above operation was repeated twice. 30 μl of DEPC treated water (RNase free water) was added to the column and centrifuged to separate total RNA.

The extracted total RNA was confirmed by agarose formaldehyde gel electrophoresis. 5 μl of 10X MOPS buffer, 9 μl of 12.3M formaldehyde, and 25 μl of formamide were added to 11 μl of total RNA and treated for 15 minutes at 55 ° C. for 1% agarose formaldehyde gel (0.7 g agarose, 10X MOPS). 7 mL of buffer, 12.3 mL of 2.3 M formaldehyde, 50.4 mL of DEPC-water) were electrophoresed. After washing for 20 minutes in 0.5M ammonium acetate and treated with 0.5M ammonium acetate containing EtBr (0.5µg / ml) for 40 minutes, total RNA was confirmed by UV transilluminator.

Generally, brain, heart, liver, lungs, spleen, etc. are used to separate total RNA from tissues. In particular, liver and spleen can separate total RNA more easily than other tissues, and yield is high. In the present invention, total RNA was isolated from the liver (rabbit) and the spleen (deer and dog) (Fig. 2). Ribosomes in eukaryotic cells containing RNA are larger and more complex than prokaryotic cells and are about 80S in size. They exist in two kinds of subunits (average 60S and 40S), with 60S having 18S rRNA and 40S with 5S, 5.8S, 28S. As a result of agarose formaldehyde gel electrophoresis of the total RNA of deer, dog, and rabbit isolated in the present invention, 28S rRNA (5000 nucleotide) and 18S rRNA (2000 nucleotide) are found among RNAs in which a large amount of eukaryotic cells are present. Confirmed.

Second step. RT-PCR Amplification of Deer, Dog and Rabbit Total RNA

RT-PCR was performed using KS1E, KS-Eco (manufactured by Seoul Bionics) primer and one-step RT-PCR kit (QIAGEN, Cat.No. 210210, USA) to amplify cDNA of deer, dog and rabbit lactoferricin. It was.

Lactoferricin cDNA was amplified by RT-PCR using the total RNA isolated from the tissues of deer, dog and rabbit as a template. RT-PCR (DNA thermal cycler, Perkin-Elmer Cetus Instruments) was run under conditions of 50 ° C 30 minutes 1cycle, 95 ° C 15 minutes 1cycle, 94 ° C 1 minute 62 ° C 1 minute 72 ° C 1 minute 30cycle, 72 ° C 10 minutes 1cycle. .

The degenerate primers KS1E and KS-Eco used in this case were cysteine that could form a disulfide bridge at the N-terminal side where lactoferricin such as humans, cattle, goats, pigs, mice, buffaloes and horses were already known. It was newly produced by referring to homology based on residues. The base sequences of the KS1E and KS-Eco primers are as follows.

Sequences of KS1E and KS-Eco Primers

KS1E primer: 5'-GGCTCGAGAAAAGACTTGGACTGTGTCTGGCT-3 '

KS-Eco primer: 5'-GGGAATTCTTAATCM ^* AGGGTCACAGCATC-3 '

* M (A, C)

When PCR-amplified the sequence of lactoferricin with KS1E and KS-Eco primers, human lactoferricin was amplified by 226bp, bovine and goat lactoferricin by 223bp, and pig lactoferricin by 217bp. It is done. As a result of RT-PCR amplification of deer, dog, and rabbit carried out in the present invention, the concentration was slightly blurred, but all appeared about 220bp size band, and the portion was estimated to be lactoferricin and used for subcloning (FIG. 3). .

Example 2 Construction of Recombinant Vector and Sequencing of Lactoferricin Gene

First step. Preparation of Lactoferricin cDNA and pPIC9 Expression Vectors

The deer, dog and rabbit lactoferricin RT-PCR products obtained by the RT-PCR amplification were electrophoresed on a 2% agarose gel. The concentration of the band was blurred, and the second PCR (95 ℃ 5 minutes 1cycle, 94 ℃ 1 minute 62) was carried out by using DNA extracted from the elution (GENOMED, JETsorb Gel Extraction kit, cat.110150) as a template. 1 cycle, 72 DEG C, 1 minute, 26 cycles, 72 DEG C, 10 minutes, 1 cycle). The 2nd PCR product was subjected to 2% agarose gel electrophoresis, followed by extraction of a 220bp band to obtain insert DNA of deer, dog, and rabbit lactoferricin.

PPIC9 vector was isolated from E. coli Top10F 'for use as expression vector of subcloning. E. coli Top10F ′ (pPIC9) with pPIC9 was plated in LB medium (10 mL) to which ampicillin (50 μg / mL) was added and incubated at 37 ° C. until late-log phase. To the cell pellet obtained by centrifugation, 1 ml of GTE buffer (50 mM glucose, 25 mM Tris pH8.0, 10 mM EDTA pH8.0) was added and mixed, followed by NS buffer (0.2N NaOH, 1% SDS). 1.5 ml each (inversion mixing, left on ice for 5 minutes). 1.5 ml of 3M potassium acetate (pH4.8) (inversion mixing, left on ice for 3 minutes), centrifugation (10,000 g, RT, 10 minutes), and only the supernatant was taken and treated with RNase (20 µg / ml, 37 ° C). , 20 minutes). An equal amount of chloroform was added and mixed (30 seconds, inverting mixing), followed by centrifugation (10,000 g, RT, 10 minutes). An equal amount of 100% isopropanol was added to the supernatant, mixed and centrifuged (12,000 g, RT, 10 minutes) to obtain pellets.

After washing with 70% ethanol and drying the pellet was added DW 32μl, 4M NaCl 8μl, 13% PEG800040μl to melt the pellet and left for 20 minutes. The pellet obtained by centrifugation again (12,000 g, 4 ° C., 15 minutes) was washed with 70% ethanol, dried, dissolved in 20 μl distilled water and electrophoresed on 0.8% agarose gel to confirm the expression vector pPIC9.

Second step. Ligation of Lactoferricin and pPIC Expression Vector DNA

The deer, dog, and rabbit lactoferricin (LFc) gene and DNA of pPIC9 were treated with Xho I for 10 hours at 37 ° C. and then treated with EcoR I for 5 hours to LFc _{-XhoI / EcoRI} , pPIC9 _{-XhoI / EcoRI} Acquired and electrophoresed in 1.2% agarose gel and extracted (JETsorb Gel Extraction kit, GENOMED, cat. 110150).

The molar concentrations of LFc- _{XhoI / EcoRI} and pPIC9- _{XhoI / EcoRI} DNA were adjusted to about 6: 1 and mixed with T4 DNA ligase and 10 × buffer for 12 hours at 16 ° C.

Third step. To check the license E.coli Transformation with DH5α

In order to confirm the ligation, E. coli DH5α competent cells were transformed.

Incubate E. coli DH5α to the mid-log phase (OD ₆₀₀ = 0.25-0.4) in LB medium supplemented with ampicillin (ampicillin, 50µg / ml) and centrifuge using pre-chilled tubes (8,000g, 4 Cells were harvested). The collected cells were mixed with ice-coldCaCl ₂ / glycerol buffer (60mM CaCl ₂ , 10mM Pipes pH7.0, 15% glycerol) and centrifuged and treated with CaCl ₂ / glycerol buffer. Centrifuged again (8,000g, 4 ℃, 5 minutes) and the cells obtained by mixing with ice-cold CaCl ₂ / glycerol buffer 100μl aliquot, and stored at -70 ℃ to prepare a competent cell.

10 μl of the mixture (LFc, pPIC9 DNA, T4 ligase, 10 × buffer) ligated at 16 ° C. was added to 100 μl of the competent cells stored at −70 ° C., and left for 20 minutes on ice. Transformation was performed by heat shock treatment at 42 ° C. for 100 seconds. After leaving it on ice for 10 minutes, 1 mL of LB medium was added, incubated at 37 ° C for 1 hour, and 100 μl of each plate was spread on ampicillin-added (20 µg / mL) LB agar plate to observe colony formation. As a result, hundreds of colonies were formed each. Lactoferricin-amplified KS1E primers contain Xho I site and KR-Eco primers contain EcoR I site. This sticky end not only increases the efficiency of ligation by ligase, but also has the advantage of requiring a lower concentration of DNA than the blunt end. In addition, different restriction enzymes are located at both ends of the amplified lactoferricin, reducing the probability of self-ligation. Indeed, self-ligation in the present invention could be confirmed by PCR amplification.

In order to confirm whether the lactoferricin gene of deer, dog, and rabbit, which is the target gene of the present invention, is ligated to the target expression vector, pPIC9, each of ten hundreds of colonies generated in LB + amp (20 µg / mL) agar plate Was inoculated in 3 ml of LB + amp (20 µg / ml) broth and incubated at 37 ° C for 10 hours to extract plasmids. The extracted plasmid was PCR amplified with 3 ＇AOX1, KS1E, and KS-Eco primers to confirm ligation.

Nucleotide sequence of 3'AOX1 primer

3'AOX1 primer: 5'-GCAAATGGCATTCTGACATCC-3 '

PCR amplification using the KS1E and KS-Eco primers used for RT-PCR confirmed that the concentration of DNA was amplified at around 220 bp, the size of the inserted lactoferricin (FIG. 4). Looking at the 10 colonies we identified, the deer had eight successes, nine dogs, and seven rabbits, which had a high ligation probability of about 80%, and self-ligation did not appear.

The newly constructed recombinant plasmids are ligated by the sticky ends of the different restriction enzymes Xho I and EcoR I at the 5 and 3 ends, so theoretically, there is no need to check the orientation of the ligation. However, in the present invention, from the standpoint of reconfirmation of the ligation, it was re-amplified using 3 ′ AOX and KS1E primers (FIG. 5). In the forward direction, a band of 374bp is expected, and if it is ligated in the reverse or wrong position rather than in the forward direction, a band of 154bp or a band other than 374bp will be expected. Fortunately, all 220bps of the amplification using KS1E and KS-Eco primers were PCR amplified into a 374bp size band and confirmed that they were forward. The recombinant vector constructed by inserting the lactoferricin gene of deer into pPIC9 was constructed with pKSSI, the recombinant vector constructed by inserting the dog lactoferricin gene into pPIC9 was constructed with pKSDO, and the rabbit lactoferricin gene was inserted into pPIC9. One recombinant vector was named pKSRA and its construction and cleavage maps are shown in FIGS. 6, 7 and 8, respectively.

Fourth step. DNA and amino acid sequence analysis of the lactoferricin gene

Recombinant plasmids pKSSI, pKSDO, and pKSRA containing deer, dog, and rabbit lactoferricin genes identified by PCR using KS1E, KS-Eco, and 3＇AOX primers were commissioned by the Institute of Basic Science in Daejeon for plasmid sequencing. Sequencing was requested and analyzed using BLAST database, DNASIS (Sequence analysis software, Hitachi, Japan). As a result, the DNA sequence of the lactoferricin site of pKSSI, pKSDO, and pKSRA and the amino acid sequence derived from the DNA are shown in FIG.

In addition, cattle, humans of which the sequence has already been identified [Mamoru Tomita, Mitsunori Takas, Hiroyuki Wakabayashi, Wayne Bellamy, antimicrobial peptides of lactoferrin. Lactoferrin: structure and function 209-218 (1994)], chlorine [Le Provos F., Nocart. M., Guerin. G. and Martin. P., Biochem. Biophys. Acta 203: 1324-1332 (1994)], E. Ashwani K. Sharma, M. Paramasivam, A. Srinivasan, MP Yadav, TP Singh J. Mol. Biol . 289: 303-317 (1998)], pigs [Margot W. Webbens, Eric D. Roush, Carlos M. Decastro, Carol A. Fierke, Biochemistry 36: 4327-4336 (1997)], camels, foxes (BLAST database) Homology with the lactoferricin amino acid sequence of the back and the like was compared (Fig. 10).

The lactoferricin gene amino acid sequence of deer, dog, and rabbit newly disclosed in the present invention has a high homology of about 60% with that of other animals. In particular, rabbit lactoferricin shows a homology of 98.5% due to the difference between the highly studied bovine lactoferricin and one amino acid (Gln → Arg). Embryonic similarities between deer, rabbits, cows, and goats appear, and the dog's lactoferricin has the same phylogeny as that of a horse.

Cationic peptides have been considered to be an important factor in many studies to investigate the antimicrobial mechanism of lactoferricin. Cationic residues form an electrical interaction with the negative charge of the micelle headgroup of the microbial membrane [Hancock REW, Falla T, Brown M. Adv Micro Rev 37: 135-175 (1995)] . For this reason cecropin [Lee, JY, Boman, A., Sun, CX, Anderson, M., Jornvall, H., Mutt, V., Boman, HG, Proc. Natl. Acad. Sci. USA . 86; 9159-9162 (1989) and magainin [Zasloff, M., Proc. Natl. Acad. Sci. USA 84, 5449-5453 (1987)], a protein containing a large amount of cationic peptides can more easily bind to the cell membranes of microorganisms and destroy the membranes. This hypothesis is based on several studies examining the antimicrobial activity of fragments chemically synthesized from Bovine Lactoferricin (BLFc) [Chantaysakorn P., Richter RL., J. food Prot 63 (3): 376-380 ( 2000), the persuasiveness was recognized by showing higher antimicrobial activity than that of small lactoferricin, which contains a large number of cationic peptides.

Therefore, lactoferricin of deer, dogs, and rabbits, which have been identified through the present invention, also contain 13, 14, and 15 cationic peptides, and this part is assumed to play a large role in antimicrobial activity. In particular, as mentioned above, rabbit lactoferricin has more cationic peptide by replacing arginine with glutamine of bovine lactoferricin, which is thought to contribute to excellent antimicrobial activity.

5th step. Secondary Structure Analysis of Deer, Dog, and Rabbit Lactoferricin Proteins

The amino acids of deer, dog, and rabbit lactoferricin identified above were analyzed by algorithms of DNASIS (Sequence analysis software, Hitachi, Japan), and the secondary structure of each lactoferricin protein was analyzed.

Looking at the helical wheel structure of deer, dog, and rabbit lactoferricin, the cationic amino acids are oriented in one place, so that they have a strong (+) charge and that hydrophilic amino acids are generally located on the right side of the wheel structure. 11, 12, and 13. That is, like other lactoferricin, deer, dog and rabbit lactoferricin has a cationic amphiphatic structure. The amphiphatic structure is shown in α-helix [Gesell J, Zasloff M, Opella SJ, J. Biomol NMR 9: 127-135 (1997)], β-sheet [Hwang PM, Zho N, Shan X, Arrowsmith CH, Vogel HJ, Biochemistry 37: 4288-4298 (1998), turn structure [Van den hooven H, Doeland CCM, Van de Kamp M, Konings RNH, Hilbers CW, Van de Ven, FJM. Eur J. Biochem 235: 382-393 (1996)] or extended / random coil structures [Schibli DJ, Hwang PM, Vogel HJ, FEBS Lett 446: 213-217 (1999)]. . The cationic amphiphatic structure also inserts a hydrophobic surface into the hydrophobic core of the micelle and cationic residues electrically interact with the negative charge of the micellar head to destroy the membrane and normal ionic gradient around the membrane. It has been reported to have antimicrobial activity by interfering with (ion-gradient) [Hwang PM, Vogel HJ. Biochem Cell Biol 76: 235-246 (1998).

According to the Hopp & Woods algorithm, the hydrophobicity of deer, dog and rabbit lactoferricin was 0.10, 0.33 and 0.18. Since hydrophobic amino acid binds to LPS more easily than hydrophilic amino acid, the high hydrophobicity of deer, dog and rabbit lactoferricin is thought to contribute to the antimicrobial activity.

Example 3 Preparation of a Transformant Incorporating a Lactoferricin Gene

DNAs of the recombinant vectors pKSSI, pKSDO, and pKSRA of the present invention were transformed into Pichia Pastoris GS115 strain by electrophoration by restriction enzyme Sal I treatment. The Sal I site is present in the backbone pKSSI, pKSDO, and pKSRA, but not on the lactoferricin structural genes of deer, dogs, and rabbits. When cleaved with Sal I restriction enzymes, pKSSI, without damage to lactoferricin, Making pKSDO and pKSRA linear makes integration easier into the genome of Peachia. When the actual attempt to transformation in the present invention, in a circular non-treated Sal I hangyeongwoo than introducing a linear processes Sal I was able to produce a more transformants.

First step. Preparation of Competent Cells

Centrifugation (1,500g, 4 ℃) by culturing Pichia pastoris GS115 strain lacking the histidine dehydogenase activity, OD ₆₀₀ = 1.3-1.5 (30 ℃, YPD 400mL) , 5 minutes), the obtained cells were washed with 200 mL ice-cold steriled DW, and the cells were mixed with 16 mL of ice-cold 1M sorbitol (centrifugal separation) (1,500 g, 4 ° C, 5 minutes) and finally Resuspension in 800 μl ice-cold 1M sorbitol was used to dispense 100 μl and then stored at −70 ° C. to prepare competent cells for transformation.

Second step. Transformation by Electrophoresis

Ice-cold 0.2 cm electroporation cuvette (INVITROGEN) of 20 μg of pKSSI, pKSDO, pKSRA and pPIC9 DNA treated with the Pchia Pastoris GS115 competent cells and restriction enzyme Sal I stored at −70 ° C. And left for 5 minutes on ice. After electroporation using electroporator II (INVITROGEN.cat.S1670-02) under conditions of charging voltage 1500V, capacitance 50㎌, resistance 200 후, 800 μl of ice-cold 1M sorbitol was added and mixed well. 200 μl of this was spread on an MD agar plate and incubated at 30 ° C. for 2 days.

Third step. His ⁺ Selection of transformants

His ⁺ transformants were observed according to the presence or absence of colonies after plating the mixture transformed by the electroporation method on the MD agar plate.

Dehydrogenase histidine (histidine) gene is a histidine requirement strain of blood Chiapas pastoris GS115 (his ^-) mutant ^{[Pichia pastoris GS115 (his -)} ] can not grow in medium lacking histidine. However, the transformants have HIS4 genes in pKSSI, pKSDO, and pKSRA so that they grow on both MD agar medium, a minimal medium lacking histidine, and MM agar medium using methanol as sole carbon source. In the present invention, strains having lactoferricin of deer, dog and rabbit among the transformants grown on both MD and MM agar medium were named Pichia pastoris SLCS, Pichia pastoris SLCD, and Pichia pastoris SLCR, respectively. Dog, SLCR obtained eight transformant colonies. At this time, the transformed pPIC9 vector into Pchia pastoris was named SLCP.

Fourth step. Mut using PCR ⁺ And Mut ^s Identification of strain

The His ⁺ transformants were incubated in 10 mL of YPD for 10 days at 30 ° C., and then genomic DNA was extracted by rapid isolation of yeast chromosomal DNA method.

PCR was performed by mixing 30ng of each transformant DNA and 10pmole of 5'AOX1 and 3'AOX1 primers in AccuPower ^TM PCR Premix (BIONEER. Cat.K-2010) (94 ° C 5 min 1cycle, 94 ° C 1 min, 55 1 minute, 72 ℃ 1 minute 25 cycles, 72 ℃ 7 minutes 1 cycle) and then Mut ⁺ , Mut ^s strain on the 0.8% agarose gel was confirmed.

Nucleotide Sequences of 5'AOX1 and 3'AOX1 Primers

5'AOX1 primer: 5'-GACTGGTTCCAATTGACAAGC-3 '

3'AOX1 primer: 5'-GCAAATGGCATTCTGACATCC-3 '

There are two phenotypes in which the recombinant vectors pKSSI, pKSDO, and pKSRA are inserted into the host Pchia pastoris G115 genome. First, the plasmid of the present invention constructed with the loss of AOX1 in the pitchia genome is replaced. At this time, due to the damage of the AOX1 gene, the transformant is deteriorated in methanol metabolism, indicating Mut ^s (methanol utilization slow). Second, the integration occurs between the HIS4 gene of the Peachia and the HIS4 gene of the plasmid. The AOX1 gene of Peachia is intact and forms Mut ⁺ (wild type for methanol utilization).

In the present invention, the deer, dog, and rabbit lactoferricin are present between the signal sequence of the expression vector pPIC9 and the 3 ′ AOX1 gene, so that the alcohol oxidase (AOX) gene is expressed during the metabolism of methanol by Peachia pastoris. Ferricin is expressed. Therefore, in order to express a large amount of lactoferricin, it is necessary to select Mut ^{+ which} is excellent in methanol metabolism among His ⁺ transformants.

When amplified His ⁺ transformants were amplified using 3`AOX1 and 5`AOX1 primers, Mut ⁺ amplified the AOX1 gene in the pitchia genome, resulting in a 2.2 kb band and pOXSI, pKSDO, and pKSRA AOX1 genes. Amplified 713 bp bands appear, but in Mut ^s , only the 713 bp band is amplified due to the loss of the AOX1 gene in the pitchia genome.

In the present invention His using 3`AOX1 and 5`AOX1 primers⁺As a result of amplifying the transformants, three of SLCS and SLCD and seven of SLCR were Mut.⁺(FIGS. 14, 15 and 16).

Pichia pastoris SLCS, Pichia pastoris SLCD and the transformants of the present invention prepared by introducing the recombinant vectors pKSSI, pKSDO, and pKSRA constructed by inserting the deer, dog, and rabbit lactoferricin genes into the Pchia pastoris GS115 strain, respectively. It was named Pichia pastoris SLCR and deposited with KCTC on February 09, 2001 with the deposit numbers KCTC 0956BP ( Pichia pastoris SLCS), KCTC 0954BP ( Pichia pastoris SLCD) and KCTC 0955BP (Pixia pastoris SLCR).

Example 4 Expression of Lactoferricin Genes in Deer, Dog, and Rabbit

First step. Transformants of the Invention Pichia pastoris SLCS, Pichia pastoris SLCD and Pichia pastoris Culture of SLCR Strains

Pre-incubation of His ⁺ transformant colonies of Pichia pastoris SLCS, Pichia pastoris SLCD and Pichia pastoris SLCR strains formed on MD agar plates at 30 ° C. and 150-200 rpm, respectively, from 10 ml of BMGY to OD ₆₀₀ = 2.0-6.0 After centrifugation (3,000 rpm, RT, 5 minutes), only cells were collected and main cultured in 100 ml of BMEY in a 500 ml baffle flask. 4 mL of 100% ethanol was induced at intervals of 24 hours from 0 hours in the main culture.

Second step. Antimicrobial Activity of Lactoferricin Against Various Microorganisms

The antibacterial activity of the expressed deer, dog, and rabbit lactoferricin was sampled by 100 μl before induction every 24 hours after the initial ethanol induction, and 70 μl of the supernatant was confirmed by paper disc method in E. coli JM109. Antimicrobial activity was measured against various strains such as Gram-positive [Gram (+)] and Gram-negative [Gram (-)] bacteria and fungi using the culture medium. At this time , Bacillus subtilis, Streptococcus mutans KCTC 3065, Streptococcus bikinensis, Listeria monocytogenes, Enerobacter aerogenos KCTC 2190, Escherichia coli JM109 KCTC 2427 , Salmonella typhimurium ATCC 19430, Eschericia coli, 0157; H7 ATCC93890, Saccharomyces serevisiae YHP 250, Pichia pastoris KCTC 7190 and the like were used. The measurement results are shown in Table 3.

Antimicrobial Spectrum Measurement of Lactoferricin of Pichia pastoris SLCS, Pichia pastoris SLCD and Pichia pastoris SLCR of the Invention Expressed by Ethanol Induction Strain Deer Lactoferricin Dog lactoferricin Rabbit Lactoferricin Gram-positive Bacillus subtilis +++ +++ +++ Streptococcus mutans ++ ++ ++ Streptomyces bikinensis - - - Listeria monocytogenes ++ ++ ++ Gram-negative Enterobacter aerogenos + + + Escherichia coli JM109 +++ +++ +++ Salmonella typhimurium +++ +++ +++ Escherichia coli O157; H7 ++ ++ ++ Yeast (Yeast) Saccharomyces cerevisiae - - - Pichia pastoris - - - [Note] Growth inhibition was scored with + when inhibition halo was visualized.

The antimicrobial spectrum of lactoferricin of Pichia pastoris SLCS, Pichia pastoris SLCD and Pichia pastoris SLCR expressed by ethanol induction showed high antibacterial activity against various bacteria and antibacterial activity against Pichia pastoris . It can be seen that Pchia pastoris was a strain suitable for the present invention. There was no significant difference in antibacterial activity between deer, dog and rabbit lactoferricin.

Third step. SDS-PAGE and Western Blotting Analysis for Expression of Lactoferricin Expressed from the Transformant

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to confirm the expression patterns and expression levels of lactoferricin in deer, dogs, and rabbits expressed from the transformants Pichia pastoris SLCS, Pichia pastoris SLCD, and Pichia pastoris SLCR. Western blot assay was performed.

Separating gel is used to achieve a concentration of acrylamide of 20% (15 mL; 30% acrylamide / 0.8% bis-acrylamide 10 mL, 0.5M Tris-HCl buffer (pH8.8) 3.75 mL, 10% SDS 150 μl, 10 % APD 50 μl, TEMED 10 μl, H ₂ 0 1 mL) and stacking gel was used to obtain acrylamide concentration of 4% (5 mL; 30% acrylamide / 0.8% bis-acrylamide 650 μl, 1.5 M Tris-HCl buffer (pH6) .8) 1.25 mL, 50 μl of 10% SDS, 25 μl of 10% APS, 5 μl of TEMED, 3 mL of H ₂ O 3). SDS-PAGE buffer was used 25mM Tris-base, 192mM glycine, 0.1% SDS, pH8.3. The culture supernatant showing antimicrobial activity was lyophilized, concentrated 10X, and precipitated with 10% TCA (trichloroacetic acid), followed by 5 × sample buffer (60mM Tris-HCl pH6.8, 25% glycerol, 2% SDS, 14.4mM 2-mer). Captoethanol and 0.1% bromophenol blue) were mixed and heat-treated at 100 ° C. for 5 minutes. The electrophoresis device was Bio-Rad, Mini-Protean II apparatus, and Bio-Rad, Kaleidoscope polypeptide standards (cat. 161-0325) were used as protein markers.

The transformant Pichia pastoris SLCS, Pichia pastoris SLCD, and Pichia pastoris SLCR culture concentrates of the present invention were subjected to SDS-PAGE (20% acrylamide gel) as described above, and then the gel was nitrocellulose membrane (Schleicher & Schuell). , Cat. 10401396) (15V, 10 hours). The membrane was treated with 2% BSA (Bovine Serum Albumin) for 2 hours and blocked, followed by washing with PBST (Phosphate Buffered Saline Tween) (twice rinse, once every 15 minutes, twice every 5 minutes). 10 μl of primary antibody was diluted in 10 mL of PBST, and the membrane was added and reacted with a slow rocking shaker for 1 hour, followed by washing with PBST. The membrane was reacted with a secondary antibody (peroxidase labeled anti-mouse antibody) diluted 1000-fold in PBST for 1 hour and washed again with PBST. Western blotting was detected using an ECL kit (amersham pharmacia biotech, RPN2108). Antibodies were prepared from a bovine lactoferricin (BLFc) protein purified as a monoclonal antibody (monoclonal antibody) and donated from Hokkaido University in Japan.

As a result of SDS-PAGE, it was confirmed in FIG. 17 that proteins of various sizes were expressed from each strain. In addition, Western blot analysis using a bovine lactoferricin fragment as a positive control revealed a protein of 7.26 KDa, the expected size of lactoferricin. In lanes 2 and 4 of the western blot, bands of 14.4 KDa, 21.7 KDa, 43.5 KDa, and 65.3 KDa were detected. This is due to the formation of two or more lactoferricin complexes by the sulfides of cysteine residues, which formed disulfide bonds in the expressed lactoferricin, in combination with the sulfur of other adjacent lactoferricin. It is estimated. Lane 3 was low in expression and most of the lactoferricin was present as a complex. Bellamy W. has reported that disulfide bonds in lactoferricin do not affect antimicrobial activity [Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K, Tomita M, Biochim Biophys Acta 1121: 130-136 ( 1992). Therefore, the modification of the lactoferricin into the complex by the cysteine residue does not affect the antibacterial activity of the lactoferricin expressed from the transformant of the present invention. In addition, the amount of lactoferricin expression was estimated to be approximately 80ng / L compared to the positive control.

As described in the above embodiments, the present invention isolates the gene encoding the lactoferricin protein having antimicrobial activity, antiviral activity and inflammatory response control activity from tissues of deer, dogs and rabbits and analyzes its DNA and amino acid sequences. Then, by inserting it into the pPIC9 subcloning vector to construct a recombinant expression vector for the expression of the recombinant lactoferricin gene and introducing it into the Pchia pastoris GS115 yeast strain to prepare a transformant expressing lactoferricin, It is effective to obtain a large amount of lactoferricin protein easily using a transformant, and since the recombinant lactoferricin shows an excellent antibacterial effect against various bacteria, yeasts and fungi strains, it is used in medicine, dairy industry, food, cosmetics and It is a very useful invention in the feed industry.

Claims (17)

The base sequence of SEQ ID NO: 1 encoding lactoferricin derived from deer. An amino acid of SEQ ID NO: 2 encoding lactoferricin from deer. The base sequence of SEQ ID NO: 3 encoding lactoferricin from dogs. An amino acid of SEQ ID NO: 4 encoding lactoferricin from dogs. The base sequence of SEQ ID NO: 5 encoding lactoferricin derived from rabbit. An amino acid of SEQ ID NO: 6 encoding lactoferricin from rabbits. A recombinant vector pKSSI constructed by inserting a gene encoding lactoferricin derived from deer according to claim 1 into an expression vector pPIC9. A recombinant vector pKSDO constructed by inserting a gene encoding lactoferricin derived from a dog according to claim 3 into an expression vector pPIC9. A recombinant vector pKSRA constructed by inserting a gene encoding lactoferricin derived from rabbit according to claim 5 into an expression vector pPIC9. The transformant Pichia pastoris SLCS, characterized in that the recombinant vector pKSSI of claim 7 constructed by inserting a deer-derived lactoferricin-encoding gene into the expression vector pPIC9 was introduced into Pichia pastoris GS115 yeast strain. KCTC 0956BP). The transformant Pichia pastoris SLCD characterized in that the recombinant vector pKSDO according to claim 8, which was constructed by inserting a dog-derived lactoferricin encoding gene into the expression vector pPIC9, was introduced into the Pichia pastoris GS115 yeast strain and transformed. KCTC 0954BP). The transformant Pichia pastoris SLCR, characterized in that the recombinant vector pKSRA according to claim 9, constructed by inserting a rabbit-derived lactoferricin-encoding gene into the expression vector pPIC9, was introduced into the Pichia pastoris GS115 yeast strain and transformed. KCTC 0955BP). A recombinant lactoferricin protein, characterized in that it is expressed from the transformants Pichia pastoris SLCS, Pichia pastoris SLCD or Pichia pastoris SLCR as described in claims 10-12. 12% recombinant yeast strain Pichia pastoris SLCS as described in claim 10, recombinant yeast strain Pichia pastoris SLCD as described in claim 11 (KCTC 0954BP) or recombinant yeast strain Pichia pastoris SLCR as described in claim 12 (KCTC 0955BP). After 24 hours of incubation in a glycerol feed medium, pre-induction with 0.5% ethanol for 4 hours, followed by introduction of 1% to 4% of 99% ethanol and fermentation culture at 26-30 ° C. for 72 hours. Ferricin production method A composition for livestock feed, food, medicine or cosmetics comprising deer lactoferricin expressed in the recombinant yeast strain Pichia pastoris SLCS (KCTC 0956BP) according to claim 10 as an active ingredient. A composition for livestock feed, food, medicine or cosmetics comprising dog lactoferricin expressed in the recombinant yeast strain Pichia pastoris SLCD (KCTC 0954BP) according to claim 11 as an active ingredient. A composition for livestock feed, food, medicine or cosmetics comprising rabbit lactoferricin expressed in the recombinant yeast strain Pichia pastoris SLCR (KCTC 0955BP) according to claim 12 as an active ingredient.