KR100383436B1 - Recombinant fungi strain and method for production of human lactoferrin antibacterial polypeptide derived from human thereof - Google Patents

Abstract

The present invention relates to a recombinant fungal strain for expressing human-derived lactoferrin and human-derived lactoferrin antimicrobial polypeptides, and recombinant human lactoferrin and human lactoferrin antimicrobial polypeptides obtained therefrom, and the present invention relates to recombinant vector pFGHLF and human-derived lactoferrin genes. The present invention recombinant fungi strains Aspergillus oryzae MMS-1 (Accession No. KCTC 0683BP) and Aspergillus oryzae MLS60 (Accession No. KCTC 0684BP) transformed by the recombinant vector pGLC into which the lactoferrin antimicrobial polypeptide gene was introduced were excellent in antibacterial activity. There is an excellent effect of mass production of recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptides.

Description

Recombinant fungi strain and method for production of human lactoferrin antibacterial polypeptide derived from human

The present invention relates to recombinant fungal strains for expressing human-derived lactoferrin and human-derived lactoferrin antimicrobial polypeptides and recombinant human lactoferrin and human lactoferrin antimicrobial polypeptides obtained therefrom. More specifically, the present invention relates to recombinant fungal strains Aspergillus oryzae MMS-1, Aspergillus oryzae MLS60 transformed by a vector recombinant with a lactoferrin gene derived from humans and a lactoferrin antimicrobial polypeptide, and recombinant human lactoferrin obtained by culturing the same. It relates to a recombinant human lactoferrin antimicrobial polypeptide.

Recently, due to the misuse, abuse, and persistence of antibiotics, which have become a major problem in the livestock and food sectors, antibiotics remain in livestock and foods, and the resistance thereof is emerging as a serious social problem. Therefore, there is an urgent need for antibiotic replacement materials that can enhance the stability of livestock products and at the same time ensure the productivity of livestock products. In addition, there is a growing demand for natural antibiotics that can replace the existing antibiotics used for the treatment of various diseases caused by bacterial infections. Natural antibiotics may be useful as feed additives and food and drug substitutes.

On the other hand, lactoferrin contains nutrients and physiologically active substances that can protect lactating offspring from various bacteria or heterologous proteins. They have extensive antimicrobial activity and are important for the protection of external antigens in mucosal areas, digestive organs, colostrum, etc. Known as a component. As described above, lactoferrin and lactoferrin antimicrobial polypeptides having a wide range of antimicrobial effects are similar to the efficacy of antibiotics, and thus can solve the problem of overuse of antibiotics, resistance and residues of livestock.

For this reason, lactoferrin was mainly isolated from milk whey, but recently, many attempts have been made to mass-produce lactoferrin by genetic engineering methods. However, there has been no method for mass production of recombinant human lactoferrin and human lactoferrin antimicrobial polypeptides by introducing and culturing human-derived lactoferrin genes and human lactoferrin antimicrobial polypeptide genes into fungal strains. Therefore, the present inventors have studied to develop a human-derived antimicrobial lactoferrin and a fungal strain that produces a large amount of a human-derived antimicrobial polypeptide, which have antimicrobial activity similar to that of antibiotics. Recombinant fungal strains Aspergillus oryzae RIB40 were transformed using the recombinant expression vectors containing the recombinant expression vectors, respectively, to obtain the recombinant fungal strains Aspergillus oryzae MMS-1 and Aspergillus oryzae MLS60, and then cultured with recombinant human lactoferrin. The present invention has been completed by mass production of recombinant human lactoferrin antimicrobial polypeptides. The mass-produced recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptides are useful as natural antibiotics to replace conventional antibiotics, and the recombinant fungal strains can be bred as industrial strains to effectively produce useful metabolites, which are of great industrial value. .

Accordingly, an object of the present invention is to provide a recombinant vector pFGHLF into which a lactoferrin gene derived from human is introduced and a recombinant vector pGLC into which a lactoferrin antimicrobial polypeptide gene derived from human is introduced. Another object of the present invention is to provide a recombinant fungal strain Aspergillus oryzae MMS-1 (Accession No. KCTC 0683BP) and Aspergillus oryzae MLS60 (Accession No. KCTC 0684BP) transformed by the recombinant vector pFGHLF and the recombinant vector pGLC. Another object of the present invention is to cultivate the recombinant fungal strains Aspergillus oryzae MMS-1 and Aspergillus oryzae MLS60 to mass produce recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptides. Still another object of the present invention is to prepare a feed containing the recombinant human lactoferrin or recombinant human lactoferrin antimicrobial polypeptide and recombinant fungal strain Aspergillus oryzae MMS-1 or Aspergillus oryzae MLS60.

The object of the present invention is to selectively recombinant only the lactoferrin and lactoferrin antimicrobial polypeptide genes from the lactoferrin cDNA isolated from humans, and then insert the amplified genes into the vector pFGC33 and pTAex3, respectively, to construct recombinant expression vectors pFGHLF and pGLC. by respectively transforming a fungal strain Aspergillus oryzae RIB40 with an expression vector recombinant fungal strain Aspergillus oryzae MMS-1 and Aspergillus oryzae was used to obtain a MLS60 the cultured fermentation of recombinant fungal strain Aspergillus oryzae MMS-1 and Aspergillus oryzae MLS60 recombinant human lactoferrin and recombinant Human lactoferrin antimicrobial polypeptides were obtained, respectively. Subsequently, after examining the antimicrobial activity of the recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptide against the pathogenic microorganisms, it was achieved by preparing a feed by adding a certain amount of each additive as feed additives.

Hereinafter, the configuration and operation of the present invention.

1A and 1B are photographic diagrams of fruiting bodies formed by growing wild type Aspergillus oryzae RIB40 among host fungi used in the present invention.

Figure 2 shows a schematic diagram of the production of recombinant vector pFGHLF for recombinant human lactoferrin expression.

Figure 3 shows a schematic diagram of recombinant vector pGLC production for recombinant human lactoferrin antimicrobial polypeptide expression.

Figure 4 is a schematic diagram of the construction of the selection marker pDE argB introduced into the host fungal strain with the recombinant expression vector pFGHLF.

Figure 5 is a schematic diagram of the construction of the selection marker pMAR1 introduced into the host fungal strain with the recombinant expression vector pGLC.

Figure 6 is a photograph showing the culture of the present invention recombinant fungal strain Aspergillus oryzae MMS-1.

Figure 7 is a photograph showing the culture of the present invention recombinant fungal strain Aspergillus oryzae MLS60.

8 is a photograph showing the antimicrobial activity against the pathogenic microorganism Bacillus subtillus KCTC3055 of the culture medium obtained by culturing the recombinant fungal strain Aspergillus oryzae MMS-1 of the present invention.

9 is a photograph showing the antimicrobial activity against the pathogenic microorganism E. coli DH5-α of the culture medium obtained by culturing the recombinant fungal strain Aspergillus oryzae MMS-1 of the present invention.

10 is a pathogenic microorganism E. coli DH5-α, E. coli 0157 V ₁ V ₂ Toxic (ATCC 15491), E. coli MV1184, E. coli JM 109 of the culture medium obtained by culturing the present invention recombinant fungal strain Aspergillus oryzae MLS60 It is a photograph showing the antimicrobial activity for each.

Figure 11 is a photograph showing the antimicrobial activity against each of the pathogenic microorganisms Salmonella typhimurium KCCM40253, Salmonella flexmery ATCC 12022 of the culture medium obtained by culturing the recombinant fungal strain Aspergillus oryzae MLS60 of the present invention.

The present invention selects and cultivates the Aspergillus oryzae RIB40 strain lacking arginine dehydrogenase activity as a host for the production of foreign proteins: the primers correspond to human lactoferrin and human lactoferrin antimicrobial polypeptides. Selective amplification of the site by PCR: ligating the amplified human lactoferrin and human lactoferrin antimicrobial polypeptide cDNA signal sequences and modifying the human lactoferrin and human lactoferrin antimicrobial polypeptide genes to vectors pFGC33 and pTAex3, respectively. And then transformed into the E. coli DH5-α strain and transformed, and separating the recombinant plasmid pFGHLF and pGLC capable of expressing human lactoferrin and human lactoferrin antimicrobial polypeptides from the transformant: ornithine carbamoyltransfera Genes encoding oriBine carbamonyl-transferase argB genes were introduced into vectors pTAex3 and pUC118, respectively, and introduced into E.coil DH5-α and transformed to confirm the introduction of human lactoferrin genes. Obtaining a large amount of the selection marker pMAR1 to confirm the introduction of the antimicrobial polypeptide gene; Recombinant fungal strains Aspergillus oryzae MMS-1 and Aspergillus oryzae MLS60 were transformed into the host strain Aspergillus aryzae RIB40 by transforming the recombinant vector pFGHLF and the selection marker pDE · argB, the recombinant vector pGLC and the selection marker pMAR1, respectively. PCR and Southern blot confirmed that the genes of human lactoferrin and human lactoferrin antimicrobial polypeptides were introduced into the chromosomal DNA of the host strain Aspergillus oryzae RIB40 after purification of genomic DNA, and recombinant human lactoferrin and recombinant human lactoferrin produced by the recombinant fungal strains. Investigating the antimicrobial activity of the antimicrobial polypeptide against pathogenic microorganisms by inhibition ring assay; Fermenting the recombinant fungal strains Aspergillus oryzae MMS-1 and Aspergillus oryzae MLS60, respectively, to mass produce recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptides; Comprising the recombinant human lactoferrin produced by mass expression and recombinant human lactoferrin antimicrobial polypeptide is added to the preparation of the feed comprises a step of producing a functional feed.

Aspergillus oryzae RIB40, a host fungal strain used in the present invention, was distributed by Dr. Byung-Ro Lee of the University of Tokyo, Japan. The Aspergillus species is a filamentous fungi, Eumycota, Plectomycetes, and Eurotiales (目). , A fungus belonging to the Eurotiaceae family and having no sexual reproduction (incomplete generation) and a non-photosynthetic fungus with a filamentous hyphae. Mycelia take nutrients to apical growth, and the tip of the mycelia protruding into the air grows spores spontaneously to make fruiting bodies. Among Aspergillus species, Aspergillus oryzae is composed of enzymes such as α-amylase (12 g / L), glucoamylase, glucosyltransferase, lipase and pectinase. Heterologous proteins such as bovine chymosin (2.5 mg / l), human lactoferrin, plant thaumatin, Muco miehei acidic protease (3.3 g / l) In the US, it was also recognized for its stability (generally recognized as safe (GRAS)).

In the present invention, the antimicrobial activity of the recombinant human lactoferrin and human lactoferrin antimicrobial polypeptide isBacillus subtilisKCTC 3055,E. coliO157 V_OneV₂Toxic ATCC 15491,Salmonella typhimuriumKCCM 40253 andSalmonella flexmeryATCC 12022 and received by Dr. Byung-Ro Lee from the University of Tokyo, JapanE. coliWith JM109 E. coliDH5-α, E. coliMV1184 was used.

All restriction enzymes used in the examples herein were purchased from Takara. Oligonucleotide primers were purchased from Bioneer, DPYA medium and minimal medium from Difco, EDTA and PMSF from Sigma, and reagents and instruments used for protein electrophoresis were from Bio-Rad. E. coli DH5-α and strain Aspergillus oryzae RIB40 according to the present invention were used for transformation.

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

Example 1: Aspergillus oryzae With MMS-1 Aspergillus oryzae Production of Human Lactoferrin and Human Lactoferrin Antimicrobial Polypeptides Using MLS60

Process 1: Optimal Host Cell Selection for Production of Human Lactoferrin and Human Lactoferrin Antimicrobial Polypeptides

In this step, Aspergillus oryzae RIB40 strain lacking arginine dehydrogenase activity was used as the test strain. Aspergillus oryzae RIB40 is MM (Minimal Medium: 10% 10 × stock solution, 0.2% trace element solution, 1.2M sorbitol, 40% glucose, 1M MgSO _4, Agar) with PDA (Potato Dextrose Agar) or arginine Or shake culture in DPYA medium or plate culture. Host fungal strains of the present invention are as shown in Figures 1a and 1b and the fruiting body is formed.

Second Step: Amplification of Human Lactoferrin and Human Lactoferrin Antimicrobial Polypeptide Genes Using PCR

Primer as shown in Table 1 to selectively PCR amplify the entire site of lactoferrin from the human lactoferrin gene cDNA, i.e., the corresponding site of the start codon and the stop codon and the 25 amino acid residues of the site of the human lactoferrin antimicrobial polypeptide. ). Promoter glucoamylase encoding of vector pFGC33 used in human lactoferrin to construct human lactoferrin and human lactoferrin antimicrobial polypoptide genes amplified according to the conditions shown in Table 2 using the designed primers in each vector Between the genes (glucoamylaseencoding gene: glaA) was designed for the restriction enzyme Hind III site, and the vector pTAex3 used for the human lactoferrin antimicrobial polypeptide was designed for the restriction enzymes Hind III and EcoR I site.

Primers used for PCR reaction of human lactoferrin (HLF) and human lactoferrin antimicrobial polypeptide (HLFc) Sample primer Sequence vector Human lactoferrin A 5'-GGACTGTGTCTGGCT AAGCTT AAAAGAAGGAGTCTTCAG-3 ' Hind Ⅲ pFGC33 B 5'GG AAGCTT CGGTTTTACTTCCTGAG-3 ' Hind Ⅲ Human Lactoferrin Antimicrobial Polypeptide A 5'-GG AAGCTT AAAAGATACGTAAAATGCTTCCCAATGG-3 ' Hind Ⅲ pTAex3 B 5'-GG GAATTC TCAAAATCTCTTTATGCAGCTG-3 ' EcoR I

PCR reaction condition Reaction condition time Cycles Heat cycle Deaturation 94 ℃ 1 sec 25 cycles 94 ℃ 1 minute 30 seconds Annealing 50 ℃ 1 sec 50 ℃ 2 minutes Polymerization 72 ℃ 1 sec 72 ℃ 3 minutes Final extension 72 ℃ 4 minutes 30 seconds shock 4 ℃

Third Step: Construction of Recombinant Vectors for Expression

The human lactoferrin gene amplified by PCR in step 2 was treated with restriction enzyme Hind III, and similarly, pFGC33 vector was also treated with Hind III, followed by ligation (Fig. 2) and amplification using PCR in the same manner. One human lactoferrin antimicrobial polypeptide gene was treated with restriction enzymes Hind III and EcoR I, and the pTAex3 vector was also treated with the same restriction enzyme and then ligation (FIG. 3). Ligation products were transformed into E. coli DH5-α strains to obtain transformants. The pFGHLF vector (FIG. 2) and the pGLC vector (FIG. 3), which are recombinant plasmids capable of expressing human lactoferrin and human lactoferrin antimicrobial polypeptides, were isolated from the transformants.

Fourth Step: Construction of Select Markers for Co-Transformation of Human Lactoferrin and Human Lactoferrin Antimicrobial Polypeptides

As a selection marker of human lactoferrin, one of the markers used for transformation of the genus Aspergillus is pTAex3 using an argB gene having an ornithine carbamoyltransferase (OTase) coding gene. The vector was treated with restriction enzyme Xba I and another vector pDE was then treated with the same restriction enzyme and then ligated (FIG. 4). This ligation product (pDE.argB) was transformed into E. coli DH5-α to obtain a transformant. In the same way, as a selection marker of human lactoferrin antimicrobial polypeptide, ornithine carbamoyltransferase (OTase) is one of the selection markers used for transformation of the genus Aspergillus as in the case of human lactoferrin. The argB gene having a coding gene of (amplified gene) was amplified by PCR and then treated with restriction enzyme Pst I, and another vector pUC118 was also treated with the same restriction enzyme and then ligated (FIG. 5). This ligation product (pMAR1) was transformed into E. coli DH5-α to obtain a transformant.

Process 5: Recombinant Fungal Strains Aspergillus oryzae With MMS-1 Aspergillus oryzae MLS60 Manufacturing

Aspergillus transformation was carried out by co-trans formation with select marker DNA. First, spore suspension and protoplast formation, second, isolation and purification of protoplasts, third, Three steps were performed to transform the expression vector and the selection marker into protoplasts using PEG (Polyethylene Glycol) and CaCl ₂ . Recombinant vectors created by genetic recombination into a host to fungi (Aspergillus oryzae) Tilburn, J. ( Tilburn, J., GG Taylor. And RW Davies. Transformation by integration Aspergillus nidulans. Gene. Vol.26, 205-221 ( 1983)) and the transformation was carried out. Suspension Aspergillus oryzae RIB40 to be used as a host is well suspended in 2 mL of 0.1% Tween80 and placed in a YPD medium and incubated (30 to 24 hours) at mid-log phase (OD ₆₀₀ = 0.6 to 1.0) at 30 ° C. After recovering only cells from 120 ㎛, IWAKI Co. Japan), 0.1g of lysing enzyme and 20mL of TF solution I (0.27M CaCl ₂ , 0.6M NaCl) were added and reacted at 30 ℃ water shaking incubator for 2 hours. Myracloth was placed on a glass filter (20-30 µm, IWAKI Co. Japan) and the culture was poured. 5 mL of TF soutionlⅡ ｛1.2M Sorbitol, 50mM CaCl ₂ , 35mM NaCl, 10mM Tris (pH 7.5)｝ was added to the culture solution, which was allowed to stand on ice for 5 minutes, followed by centrifugation (3,000rpm, 10min, 4 ℃). pellets) were recovered, and 10 mL of TF solution II was added thereto, followed by reaction on ice for 5 minutes, followed by centrifugation to recover pellets. Thereafter, the mixture was suspended in 1 mL of TF solution II, and then 10 µl of recombinant plasmid and 10 µl of selection marker DNA were added and allowed to react at room temperature for 25 minutes. Next, TF solution III ｛60% PEG4000, 50mM CaCl ₂ , 10mM Tris (pH 7.5)｝ divided into 125µL twice, 425µL was added at the end, suspended well, reacted at room temperature for 20 minutes, and recovered by centrifugation. Was added to 500 μl of TF solution II and smeared on MM medium, and 0.6% Top agar (NO ₃ , sorbitol, glucose) was poured thereon to form a medium medium. Incubated for 4 to 5 days in a 30 ℃ incubator (incubator). The transformed transformants were cultured in DPYA medium to purify the genomic DNA of human lactoferrin and human lactoferrin antimicrobial polypeptides, and then subjected to PCR reactions using primers A and B described above and to probe human lactoferrin and human lactoferrin antimicrobial polypeptide genes. Southern blot was used to finalize the bacteria into which the human lactoferrin and human lactoferrin antimicrobial polypeptide genes were inserted. The culture of the transformed recombinant fungal strain Aspergillus oryzae MMS-1 is shown in Figure 6, the transformed recombinant fungal strain Aspergillus oryzae MLS60 is shown in Figure 7. After transforming the final transformant Aspergillus oryzae MMS-1 to OD ₆₀₀ = 0.8 to 1.7 in DPYA medium, transformants were transformed by time zones incubated on LB agar agar medium evenly spread on Bacillus subtilis KCTC 3055. Paper disks moistened with 60 μl of culture medium were incubated at 30 ° C. for 40 to 48 hours, and growth growth zones were observed. E. coli DH5-α was also formed in the same manner. As a result, it was confirmed that the inhibitory ring is formed as shown in Figure 8 and 9 that there is antibacterial activity. After incubating the final transformant Aspergillus oryzae MLS60 in the DPYA medium to OD ₆₀₀ = 0.8-1.7 by the above method, E. coli DH5-α, E. coli 0157 V ₁ V ₂ Toxic ATCC 15491 in LB agar agar medium. , Incubate paper discs moistened with 60 μl of transformant culture medium for each time period incubated on medium-layered medium in which E. coli MV1184 and E. coli JM109 were evenly spread. The formation of growth inhibition zone was observed. In the same way, antibacterial activity against Salmonella typhimurium KCCM40253 and Salmonella flexmery ATCC 12022 was investigated. As a result of the experiment, growth inhibition ring was formed as shown in FIG. 10 and FIG. 11 to confirm that there was antibacterial activity.

The recombinant fungal strain Aspergillus oryzae MMS-1 of the present invention and the recombinant fungal strain Aspergillus oryzae MLS60 were deposited on November 5, 1999, Accession No. KCTC 0683BP and Accession No. KCTC 0684BP at the Microbial Deposit Center of the Biotechnology Research Institute.

Example 2: For mass production of human lactoferrin and human lactoferrin antimicrobial polypeptides Aspergillus oryzae With MMS-1 Aspergillus oryzae Fermentation culture of MLS60

In order to determine the production of human lactoferrin of the transformant Aspergillus oryzae MMS-1 obtained in Example 1 and the production of human lactoferrin antimicrobial polypeptide of Aspergillus oryzae MLS60, the recombinant fungal strains of each of the recombinant fungi were DPYA (dextrin polypepton yeast extract ammonium sulfate). Add 0.4% (NH ₄ ) ₂ SO ₄ to pH 5.6 to make the spore-forming medium (SMM, 10ml 10 × stock solution, 0.1ml trace element solution, 2g Bacter agar, 2% starch, 3% NaCl, 0.2ml) Spores grown in 1 M MgSO ₄ ) were incubated in 50 mL of the culture with shaking at 30 ° C. for 24 hours. This is called seed culture, and baffle Erlenmeyer flask medium containing 50 mL of culture was aseptically inoculated in a 5 L fermenter to scale up from the species culture. 5L fermenter was sterilized and then sterilized with ammonia water pH 5.6 and inoculated with 50mL culture to carry out the fermenter main culture. Fermentation conditions were carried out at an air supply of 1 VVM (volume of air per volume of medium per minute), stirring speed 1200 rpm, temperature 30 ℃, pH 5.6. As a protease inhibitor, 5 mL (1mM / total volume) of 500mM EDTA, a metalloproteinase inhibitor, is added in about 25-30 hours, and a serine proteinase inhibitor After adding 5 mL (1 mM / total volume) of 100 mM PMSF (phenyl methyl sulfonyl fluoride), the culture medium and the cells were separated. The medium used in the present invention was a fungus-only complex medium PDB (Potato dextrose broth) medium, 2% soluble starch, 0.2% casamino acid in the Erlenmeyer flask medium, and DPYA [ (2% dextrin, 1% polypeptone, 0.5% yeast extract), 0.5% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.4% (NH ₄ ) ₂ SO ₄ ), 0.2% casamino acid , 2% soluble starch was used. To isolate and purify the human lactoferrin and human lactoferrin antimicrobial polypeptides, the culture medium was placed in Centriprep-50 (50KDa or more, Amicon Co. USA) and Centriprep-3 (3000Da or more, Amicon Co. USA) and 20mM phosphate buffer ( pH 8.0) was centrifuged with buffer exchange. Centrifugation was repeated three times for 30 minutes at 4,000 rpm, 4 ℃. The amount of human lactoferrin obtained by culturing in this manner was about 0.5 g / L, and the amount of human lactoferrin antimicrobial polypeptide was 0.4 g / L.

At this time, the stock solution in the spore-forming medium contained NaNO ₃ 6%, KCl 0.52%, KH ₂ PO ₄ 1.52% and pH 6.5 and the trace element solution is FeSO ₄ · 7H ₂ O 1%, ZnSO ₄ · 7H _{2 O 0.88%, CuSO 4 · 5H} 2 O 0.04%, MnSO 4 · 4H 2 O 0.015%, Na 2 B 4 O 7 · 10H 2 O 0.01%, (NH 4) 6 Mo 7 O 24 · 4H 2 O 0.005g It contains.

Example 3: Preparation of feed containing recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptide, respectively

In this embodiment, the feed using the recombinant fungal microbial strains Aspergillus oryzae MMS-1 and Aspergillus oryzae MLS60 fermentation culture in Example 2, each of the recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptide as a feed additive along with the recombinant fungal cells Prepared. Recombinant fungal strains Aspergillus oryzae MMS-1 or Aspergillus oryzae MLS60 transformed from fermented cultures into a carrier mixed with an excipient in a range of 0.05 to 1.5% of the total weight of the feed and the recombinant humans produced therefrom Lactoferrin or recombinant human lactoferrin antimicrobial polypeptides were mixed.

Or more, and it transformed the invention recombinant fungal strain Aspergillus oryzae MMS-1 by a recombinant vector pGLC a human-derived lactoferrin gene is introduced into a recombinant vector pFGHLF and human-derived lactoferrin antimicrobial polypeptide gene was introduced as described above through the embodiments Fermentation of Aspergillus oryzae MLS60 is a very useful invention for biopharmaceutical and animal husbandry because it has an excellent effect of mass production of recombinant human lactoferrin and recombinant human lactoferrin antimicrobial polypeptide having excellent antibacterial activity.

Claims (10)

delete delete delete Recombinant fungal strain Aspergillus olease (Aspergillus oryzae) MLS60 (KCTC 0684BP). delete delete Aspergillus olease according to claim 4 (Aspergillus oryzae) 0.4% (NH) after growing MLS60 (KCTC 0684BP) in spore forming medium₄)₂SO₄After incubation by shaking with overnight shaker in DPYA (dextrin polypepton yeast extract ammonium sulfate) medium, the seed culture was inoculated in a funnel culture Erlenmeyer flask medium, and the Erlenmeyer flask was incubated in a 5L fermenter to enlarge the scale. Method of producing a recombinant human lactoferrin antimicrobial polypeptide, characterized in that the culture is inoculated by inoculation. The method of claim 7, wherein the spore-forming medium is NaNO ₃ 6%, KCl 0.52%, KH ₂ PO _{4 Stock} solution containing 1.52%, FeSO ₄ · 7H ₂ O 1%, ZnSO ₄ · 7H ₂ O 0.88%, CuSO _4, trace elements containing _{5H 2 O 0.04%, MnSO 4} · 4H 2 O 0.015%, Na 2 B 4 O 7 · 10H 2 O 0.01%, (NH 4) 6 Mo 7 O 24 · 4H 2 O solution, Bacter agar and 3% NaCl, 1M MgSO ₄ , the Erlenmeyer flask medium contains PDB (Potato dextrose broth) medium, 2% water-soluble starch, 0.2% casamino acid, the fermentation tank for this culture is 2 DPYA (dextrin) containing% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.4% (NH ₄ ) ₂ SO ₄ , 0.2% casamino acid, 2% water soluble starch Polypepton yeast extract ammonium sulfate) medium, characterized in that the recombinant human lactoferrin antimicrobial polypeptide production method. delete delete