JP2003135060A - METHOD FOR PRODUCING BOVINE INTERLEUKIN-1beta - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for mass-producing high-purity bovine interleukin-1β (bIL-1β). SOLUTION: This method comprises culturing Brevibacillus choshinensis transformed by a plasmid integrated with a DNA fragment having a base sequence encoding the amino acid sequence of matured bovine IL-1βto produce and accumulate the bovine IL-1β in the culture and then collecting the bovine IL-1β.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to efficient and large-scale production of bovine interleukin-1β (bIL-1β) (hereinafter also referred to as “bovine IL-1β”) by gene recombination technology. The present invention relates to a method for producing, more specifically, culturing Brevibacillus choshinensis transformed with a plasmid incorporating a DNA of a gene encoding the amino acid sequence of mature bovine IL-1β, The present invention relates to a method for producing bovine IL-1β, which comprises collecting bovine IL-1β produced in a culture.

[0002]

2. Description of the Related Art Interleukin-1:
IL-1) is a kind of cytokine, and its existence has been known for a long time, and it has been reported as an endogenous pyrogen, lymphocyte activating factor, hepatocyte stimulating factor, etc. Are all interleukin-1 (IL-
1) was unified under the name (Cellular Immunol., 48 , 4
33 (1979)). The existence of two different genes encoding IL-1 precursors was subsequently reported (Nature, 315, 641 (1
985)), which is deduced from the nucleotide sequences of these two genes.
The presence of 1L-1α consisting of 59 amino acids and IL-1β consisting of 153 amino acids was revealed.

As the applied development of IL-1, clinical development for anticancer agents, aplastic anemia, myelodysplastic syndrome, etc., protection of side effects by anticancer agents, and adjuvants for vaccines are being studied. Recently, IL-1 has been found to be effective in the prevention and treatment of diseases such as mastitis in livestock such as cattle, sheep, and goats, and its use as an auxiliary treatment along with conventional antibiotic treatment has been studied, and it is effective. Development of various production methods is required.

[0004]

As described above, IL-
It was revealed that 1β is effective for the prevention and treatment of mastitis in livestock. Cattle I occupy an important position in the livestock industry
Studies have also been conducted on L-1β, and it was confirmed that bovine IL-1β is effective in treating mastitis in dairy cows.

This bovine IL-1β is biosynthesized as a membrane-bound precursor of 266 amino acid residues. Bovine IL-1β
113 residues from the N terminus of the precursor are a secretory signal peptide, and the 153 amino acid residue obtained by cleaving the 113 residues is secreted extracellularly as mature bovine IL-1β.
The precursors of bovine IL-1β and the amino acid sequences of mature bovine IL-1β are shown in SWISS-PROT: P09428 and the like. The amino acid sequence of mature bovine IL-1β is shown in SEQ ID NO: 1 (FIGS. 1 and 2). A large amount of bovine IL-1β is required for using this bovine IL-1β for the treatment of cattle, but it was present only in a very small amount in the living body, and there was practically no means for obtaining it. The present invention provides a method for producing bovine IL-1β in large quantities.

[0006]

The present invention has been made to achieve the above object, and bovine IL-1β is present in the living body in a very small amount. Considering that it is not a profitable method at least industrially, as a result of examining from various directions, we focused on efficient mass production by gene recombination technology.

[0007] As a result of earnest research, the present inventors have succeeded in cloning a gene encoding bovine IL-1β, have succeeded in preparing an expression plasmid containing the gene, and efficiently expressed the gene. The host strains that can be used were extensively screened from all sides. And I focused on Brevibacillus chosinensis.

Brevibacillus chosinensis ( Brev
ibacillus choshinensis ) ( formerly Bacillus brevis ). Shida O. et al., Int. J. Syst. B
Acteriol., 46, 939-946 (1996) changed the taxonomic position. ), There are many strains that secrete and produce proteins outside the cells and do not produce proteolytic enzymes in the culture broth, and are used as host strains for gene recombination in the production of various proteins. For example, Takagi et al., Brevibacillus choshinensis HPD3, which is one of the strains belonging to Brevibacillus choshinensis
1 (FERM BP-6863) (this strain is the same strain as Bacillus brevis H102 (FERM BP-1087)) was established as a host bacterium, and a mass production technology of h-EGF which is one kind of cytokine was established, and h-EGF was established. It has succeeded in secreting and producing about 3 g / l of EGF in a culture solution (BIO INDUSTRY, 8 , 275 (1991)).

The present inventors have found that Brevibacillus choshinensis HPD31-S5 (FERM BP) which is a mutant strain of this Brevibacillus choshinensis HPD31.
-6623) was used to transform a plasmid into which a DNA fragment having a nucleotide sequence encoding the amino acid sequence of mature bovine IL-1β was incorporated, and it was confirmed that a transformant was obtained. By culturing the transformant, the target DNA incorporated in the expression plasmid vector was expressed, and the bovine IL-1β was successfully secreted and produced in large quantities outside the bacterial cells. Based on these useful new findings. Further research is conducted here on bovine IL
We have completed a method for mass producing -1β.

That is, the present invention relates to mature bovine IL-1.
DNA having a nucleotide sequence encoding the amino acid sequence of β
By culturing Brevibacillus choshinensis transformed with the plasmid incorporating the fragment,
The present invention relates to a method for producing bovine IL-1β, which comprises producing and accumulating bovine IL-1β in a culture, and collecting this. Hereinafter, the present invention will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The amino acid sequence of the protein produced by the production method of the present invention is not particularly limited as long as it has bovine IL-1β activity, but preferably mature bovine IL-1β (SEQ ID NO: 1). In the present invention, the DNA having the nucleotide sequence encoding mature bovine IL-1β is represented by SEQ ID NO: 1
Any DNA may be used as long as it has a base sequence encoding the amino acid sequence of bovine IL-1β shown in (FIG. 1, FIG. 2). For example, DNA having the base sequence shown in SEQ ID NO: 6 (FIG. 1, FIG. 2) Can be used. Note that FIG.
In FIG. 2, the amino acid sequence (153 amino acids) of mature bovine IL-1β is shown in the lower row, and the nucleotide sequence (462b
p) is shown in the upper row.

The vector for introducing and retaining the DNA fragment having the nucleotide sequence encoding bovine IL-1β into a host bacterium may be any plasmid capable of replicating in the host bacterium. For example, pUB110 and pNU200 (Shigezo Utaka, Journal of the Japanese Society of Agricultural Chemistry, 61 , 669 (1987)), pHY70, which can be replicated in Brevibacillus choshinensis.
0 (S. Ebisu et al., Biosci. Biotech. Biochem., 5
6 , 812-813 (1992)), pNH300 (Shiga, Y et al., App
L. Environ. Microbiol., 58, 525-531 (1992)), pH
Examples include plasmids such as T110 (Japanese Patent No. 2727391) and derivatives thereof. As a particularly preferable example, pNH326 (Ka which is a derivative of pNH300
jino, T. et al., Appl. Environ. Microbiol., 66, 638-642.
(2000)). pNH326 is pN
The secretory signal sequence of the HWP promoter region of H300 is a modified secretory signal sequence of R2L6 type (JP-A-7-17
0984) and pHT926 (Ebisu, S. et al., Appl. Environ. Mi) downstream of the multiple cloning site.
crobiol., 61, 3154-3157 (1995)), which is a plasmid vector constructed by inserting a DNA encoding a transcription terminator sequence. As a method for constructing these plasmids, known methods are appropriately used, for example, Molecular Cloning, A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory (Molecular Cloning 2nd ed., A Laboratory).
The method described in Manual, Cold Spring Harbor Laboratory, 1989) is exemplified.

The bacterium used as a host bacterium in the present invention is Brevibacillus choshinensis HPB31 (FERM).
BP-6863) and its variant Brevibacillus
Choshinensis HPD31-S5 (FERM BP-
6623) and the like can be preferably used.

The method for transforming Brevibacillus choshinensis, which is a host bacterium, may be an electroporation method, a protoplast method, a PEG method or other known methods. For example, the method of Takahashi et al. (Takahashi et al.,
J. Bacteriol., 156 , 1130 (1983)) or the method of Takagi et al. (H. Takagi et al., Agric. Biol. Chem., 53 , 30.
99-3100 (1989)) and the like.

The medium used for culturing the transformed Brevibacillus choshinensis may be any medium as long as the transformant can grow and produce bovine IL-1β.

Examples of the carbon source contained in the medium include glucose, sucrose, glycerol, starch,
Dextrin, molasses, organic acid, etc. are used. As the nitrogen source, casein, peptone, meat extract, yeast extract, casamino acid, organic nitrogen sources such as glycine, urea,
An inorganic nitrogen source such as ammonium sulfate is used.
In addition, inorganic salts such as potassium chloride, monopotassium phosphate, dipotassium phosphate, sodium chloride and magnesium sulfate are added to the medium as needed. For auxotrophic bacteria, nutrient substances necessary for their growth may be added to the medium. Examples of the nutritional substance include amino acids, vitamins, nucleic acids and the like.

In addition, if necessary during the culture, antibiotics for the culture, such as penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine,
Add ampicillin, neomycin, etc. Further, if necessary, a defoaming agent such as soybean oil, lard oil, various surfactants and the like may be added. The initial pH of the medium is 5.0-9.
0, and more preferably 6.5 to 7.5. The culture temperature is usually 15 ° C to 42 ° C, more preferably 24 ° C to 37 ° C.
C., and the culture time is usually 16 to 166 hours, more preferably 24 to 96 hours.

In the present invention, bovine IL-1β is added to the culture by culturing the transformant under the above-mentioned conditions.
Are generated and accumulated. Bovine IL thus obtained
-1β can be purified by known methods, for example, by membrane treatment using a UF membrane, MF membrane, etc., ammonium sulfate fractionation method, chromatography and the like (basic experimental method for proteins and nucleic acids, Nankodo (1985)).

Collection and purification of bovine IL-1β may be carried out by appropriately utilizing known protein purification methods as described above, and solvent extraction, salting out, desalting, organic solvent precipitation, ultrafiltration,
Methods such as ion exchange, hydrophobic interaction, HPLC, gel filtration and affinity chromatography, electrophoresis, and isoelectric focusing can be used alone or in combination of two or more.

Specifically, for example, the following may be performed. First, the culture is centrifuged or treated with an MF membrane to remove bacterial cells, ammonium sulfate is added to the supernatant or the filtrate, and the supernatant is filtered through a UF membrane to obtain bovine IL-1β. It is also possible to carry out purification by further collecting and fractionating with a UF membrane having a different molecular weight cutoff for further purification. The fractionated fraction may be further fractionated and purified by an ion exchange resin or the like, and may be lyophilized if necessary. Regarding the physiological activity of bovine IL-1β thus obtained, the known MTT method (T. Mosmann, J. Immunol. Met
h., 65, 55-63 (1983)).

According to the present invention, Brevibacillus choshinensis can be used as a host bacterium, and bovine IL-
For the first time, it became possible to produce 1β in large quantities and outside the cells. Particularly in the present invention, bovine IL-1β
Not only was it made possible to produce a large amount by genetic recombination technology, but since bovine IL-1β is secreted and produced outside the cells, the recovery and purification steps are extremely simplified, and the risk of contamination is high. The bovine IL-1β can be produced in large quantities with high efficiency and low purity.

Hereinafter, the present invention will be described in more detail by way of examples, which are merely illustrative and the present invention is not limited thereto.

[0023]

Example 1 Preparation of Transformant (1) Cloning of bovine IL-1β gene From bovine peripheral blood, Ficoll-Conray gravity centrifuge method (Shinsei Kagaku Koza 12-I, Tokyo Kagaku Dojin, edited by The Biochemical Society of Japan, p3-6) to separate mononuclear cells (PBMCs),
LPS at 10 μg / ml for 4 hours in vitro
It was stimulated with (lipopolysaccharide). This PBMCs
Poly (A) + RNA was extracted from the above and cDNA was obtained by reverse transcriptase.

SEQ ID NO: 2 (FIG. 3) and SEQ ID NO: 3 by the PCR method using the bovine cDNA obtained here as a template.
The primer of FIG. 4 (FIG. 4) was synthesized to encode a bovine IL-1β precursor DNA (about 80
1 bp) was amplified. The amplified PCR product was ligated to the plasmid pCR II (manufactured by Invitrogen) using the TA cloning method (TA Cloning Kit manufactured by Invitrogen). This connection DN
E. coli INVαF '(manufactured by Invitrogen) was transformed with A, and LB agar medium (1.0% Tryptone, 0.5% Y) was used.
east Extract, 1.0% NaCl, pH 7.0, 1.5%
(Agar) and ampicillin resistant strains were selected. A plasmid pCRIIb carrying a DNA encoding a bovine IL-1β precursor by extracting a plasmid from the selected strain
IL-1β was obtained.

Furthermore, a report by Steven R. Leong et al. (Nucleic
Acids Res., 16 , 9054 (1988)), two kinds of primer DNAs (BIL-1-N: SEQ ID NO: 4 (FIG. 5) and IL-1-C: SEQ ID NO: 5 (FIG. 6) were synthesized.
Two types of primers B synthesized as described above using the plasmid pCRII bIL-1β obtained above as a template
It was amplified by the PCR method using IL-1-N and IL-1-C.

PCR is carried out using the primer BIL of SEQ ID NO: 4.
-1 -N and the primer IL-1-C of SEQ ID NO: 5 were each 100 pmol, Taq polymerase 2.5 unit, dN
TP200 μM, pCRII bIL-1β template DNA
1 ng, 100 μl Taq buffer solution (10 mM Tris-hydrochloric acid (pH 8.5), 2.5 mM Mg ²⁺ , 50 mM potassium chloride, 100 μg / ml bovine serum albumin) were mixed and kept at 96 ° C. for 30 seconds, and then heat denaturation of DNA (94
℃, 60 seconds), primer annealing (54 ℃, 6
0 second), and extension of the primer (70 ° C., 60 seconds) was performed for 25 cycles.

The amplified DNA fragment was digested with NcoI and Ba.
After treatment with mHI, it is subjected to 0.8% agarose gel electrophoresis and contains a nucleotide sequence (462 bp: including termination codon TAA) encoding mature bovine IL-1β.
A bp DNA fragment was recovered.

(2) Bovine IL-1β secretion expression vector p
Construction of NH326b IL-1β pNH326 (Kajino, T. et al., Appl. Environ., Microb
iol., 66 , 638-642 (2000)) with NcoI and Bam HI
And then subjected to 0.8% agarose gel electrophoresis to recover the 4.0 kb fragment, and obtain the mature bovine IL obtained above.
482 bp DNA containing a nucleotide sequence encoding -1β
The fragments were ligated with T4 DNA ligase. Connection D
Brevibacillus choshinensis HPD using NA
31-S5 (FERM BP-6863) was transformed with neomycin 50 mg / ml in TM (TMNm).
A strain having neomycin resistance was selected by plating on agar medium. Plasmids are extracted from selected strains to produce mature bovine IL
Plasmid pNH carrying DNA encoding -1β
326b IL-1β was obtained.

The strain obtained here was used as Brevibacillus choshinensis HPD31-S5 / pNH326bIL-1β ( Brevibacillu
s choshinensis HPD31-S5 / pNH326bIL-1β) and was deposited domestically as FERM P-18241 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology.

[0030]

Example 2 Culture of Transformant and Purification of Bovine IL-1β (1) Brevibacillus choshinensis HPD31-
Culture of S5 / pNH326bIL-1β Obtained transformant Brevibacillus choshinensis HPD31-S5 / pNH326bIL-1β 30 L
3 in AFNm liquid medium using jar fermenter
Shake culture was performed at 0 ° C for 2 days, and the culture supernatant was subjected to SDS.
Analysis was performed by Western blotting using PAGE and human IL-1β monoclonal antibody. As a result of the analysis, a staining band was shown at the position of the molecular weight corresponding to bovine IL-1β, and the concentration of bovine IL-1β was 200 mg /
It was confirmed that it was produced in L medium. B. cho
Shinensis HPD31-S5 / pNH326
The pattern of secretory production of bovine IL-1β by bIL-1β is shown in a photograph substituted for a drawing (FIG. 7). In the figure, CBB st
ain shows a Coomassie Brilliant Blue stained image,
W. B. Shows a Western blotting image.

(2) Purification of bovine IL-1β Brevibacillus choshinensis HPD obtained in (1)
31-S5 / pNH326b IL-1β culture medium 18L
Was sterilized using a Pellicon cassette membrane (Millipore) having a pore size of 0.45 μm to obtain about 18 L of bovine IL-1β culture supernatant. Next, 1% of bovine IL-1β culture supernatant was added.
Ammonium sulfate (1000 g) was added to 0% saturation, well dissolved, and allowed to stand for 3 hours or more. Bovine IL-1 with ammonium sulfate
The β culture supernatant was filtered using a Pellicon cassette membrane with a pore size of 0.45 μm, and bovine IL-1β was collected on the concentrated liquid side.

The recovered concentrated bovine IL-1β was added to 10,
Using a 000 fraction Pellicon cassette membrane, it was dialyzed against pure water to collect bovine IL-1β on the concentrated liquid side. Further, this recovered solution was freeze-dried. Freeze dried sample to SDS-P
AGE, Lowry method (OHLowry et al., J. Biol. Ch
Em., 193 , 265-275 (1951)), the result showed that the purified bovine IL-1β had a purity of about 50% and the recovery rate from the culture was 60%.

[0033]

Example 3 Activity Measurement of Bovine IL-1β The bovine IL-1β obtained in Example 2 was derived from a mouse cell, D10. Using G4.1 cells, the MTT method (T. Mosman
n, J. Immunol, Meth., 65, 55-63 (1983)). D10. Derived from mouse cells during storage in liquid nitrogen (-196 ° C). G4.1 cells are rapidly lysed at 37 ° C. and cryopreserved cells are well suspended in 50 ml of RPMI1640 medium (without serum). Centrifuge the cell suspension (4
℃, 1,000 rpm, 10 minutes) After removing the supernatant, RP
After resuspending in MI1640 medium (containing 20% serum), transfer to a Petri dish, and in a CO ₂ incubator at 37 ° C. for 2-3.
Incubate for a day. Furthermore, D10. RPMI of G4.1 cells
Subculture in 1640 medium containing 10% FCS (fetal calf serum). After completion of the culture, D10. G4.1 cells are peeled off from the culture dish, being careful not to crush the cells. The cell suspension obtained was measured for the number of cells using a hemocytometer and
Cells are distributed in a 6-well dish so that the concentration of each well is 100 μl / well. bIL-1β is adjusted to 500 ng / ml to 0.06 ng / ml by a two-fold dilution method, and 100 μl of each is distributed to a 96-well petri dish. 1 at 37 ° C in an incubator containing 5% CO _2.
After culturing for 8 hours, 25 μl of MT was added to each nuclear well.
T solution (MTT; 3- (4,5-dimethylthiazol-2-yl) -2,5-d
iphenyltetrazolium bromide dissolved in 5 mg / ml PBS). After culturing for 2 hours in an incubator containing 5% CO ₂ at 37 ° C., 100 μl of the culture solution was absorbed, and the elution solution (12.5% SDS (w / v), 50%
DMF (N, N-dimethylfolmamide), 2.5% acetic acid (8
0% concentration), 2.5% 1N hydrochloric acid pH 4.7) 100μ
1 / well was added. After extraction at 37 ° C. overnight, the mixture was stirred well and the absorbance was measured (OD at 570n
m). As a result, it was confirmed that the recombinant bovine IL-1β showed the same activity as the natural IL-1β obtained from bovine.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, high-purity bovine IL-1
For the first time, β can be efficiently mass-produced,
The recombinant bovine IL-1β thus obtained can be effectively used for the prevention and / or treatment of mastitis in domestic animals.

[0035]

[Sequence list]                           SEQUENCE LISTING <110> National Agricultural Research Organization;                Higeta Shoyu Co., Ltd.,; And Fujita Gakuen <120> Producing Method of Bovine Interleukin-1β <130> 6443 <141> 2001-10-31 <160> 6 <210> 1 <211> 153 <212> PRT <213> Bovine gene <400> 1 Ala Pro Val Gln Ser Ile Lys Cys Lys Leu Gln Asp Arg Glu Gln Lys   1 5 10 15 Ser Leu Val Leu Ala Ser Pro Cys Val Leu Lys Ala Leu His Leu Leu              20 25 30 Ser Gln Glu Met Asn Arg Glu Val Val Phe Cys Met Ser Phe Val Gln          35 40 45 Gly Glu Glu Arg Asp Asn Lys Ile Pro Val Ala Leu Gly Ile Lys Asp      50 55 60 Lys Asn Leu Tyr Leu Ser Cys Val Lys Lys Gly Asp Thr Pro Thr Leu  65 70 75 80 Gln Leu Glu Glu Val Asp Pro Lys Val Tyr Pro Lys Arg Asn Met Glu                  85 90 95 Lys Arg Phe Val Phe Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu Phe             100 105 110 Glu Ser Val Leu Tyr Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile Glu         115 120 125 Glu Arg Pro Val Phe Leu Gly His Phe Arg Ala Gly Gln Asp Ile Thr     130 135 140 Asp Phe Arg Met Glu Thr Leu Ser Pro 145 150 <210> 2 <211> 24 <212> DNA <213> Artificial sequence <400> 2 atggcaaccg tacctgaacc catc 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 ttagggagag agggtttcca ttct 24 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <400> 4 aaaccatggc tttcgctgca cccgttcagt caataaag 38 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 aaaggatcct tagggagaga gggtttccat 30 <210> 6 <211> 462 <212> DNA <213> Artificial sequence <400> 6 gcacccgttc agtcaataaa gtgcaaactc caggacagag agcaaaaatc cctggtgctg 60 gctagcccat gtgtgctgaa ggctctccac ctcctctcac aggaaatgaa ccgagaagtg 120 gtgttctgca tgagctttgt gcaaggagag gaaagagaca acaagattcc tgtggccttg 180 ggtatcaagg acaagaatct atacctgtct tgtgtgaaaa aaggtgatac gcccaccctg 240 cagctggagg aagtagaccc caaagtctac cccaagagga atatggaaaa gcgctttgtc 300 ttctacaaga cagaaatcaa gaatacagtt gaatttgagt ctgtcctgta ccctaactgg 360 tacatcagca cttctcaaat cgaagaaagg cccgtcttcc tgggacattt tcgagctggc 420 caggatataa ctgacttcag aatggaaacc ctctctccct aa 462

[Brief description of drawings]

FIG. 1 Amino acid sequence of mature bovine IL-1β (bottom row)
And the nucleotide sequence of the DNA encoding the same (upper row).

FIG. 2 shows the continuation of the above.

FIG. 3 shows the primer (SEQ ID NO: 2).

FIG. 4 shows a primer (SEQ ID NO: 3).

FIG. 5 shows the primer BIL-1-N.

FIG. 6 shows primer IL-1.

FIG. 7 is a drawing-substituting photograph showing a Coomassie Brilliant Blue stained image and a Western blotting image of the cultures of the transformant (2) and the non-transformant (control) (1).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Makoto Mizukami             7293-9 Hasaki-machi, Kashima-gun, Ibaraki (72) Inventor Yasuaki Murahashi             4-6-9 Higashiota, Kisarazu City, Chiba Prefecture             Ru Kazusa Po3 B-106 (72) Inventor Shogo Ebisu             2798-1 Shimizu-cho, Choshi-shi, Chiba (72) Inventor Yuichi Yokomizo             4-chome, Namiki, Tsukuba, Ibaraki 11 919-103 (72) Inventor Yasuyuki Mori             1-2, Tsukuba, Ibaraki (72) Inventor Shigeki Inumaru             4-10-1, Namiki, Tsukuba City, Ibaraki Prefecture (72) Inventor Takao Tsuji             1-98 Tarakugakubo, Kutsukake Town, Toyoake City, Aichi Prefecture             Corporate Fujita Health University, Faculty of Medicine F-term (reference) 4B024 AA01 AA10 AA11 BA26 CA04                       CA20 DA05 EA04 HA03                 4B064 AG04 CA02 CA19 CC01 CC03                       CC06 CC24 CD30 CE03 CE04                       CE06 CE14 CE17 DA01 DA11                       DA13

Claims (3)

[Claims] 1. Bovine interleukin-1β (bovine I
nterleukin-1β: bIL-1β) Brevibacillus choshinensi transformed with a plasmid incorporating a DNA fragment having a nucleotide sequence encoding the amino acid sequence of a protein having the activity.
s ) by culturing bovine interleukin-1
A method for producing bovine interleukin-1β, which comprises producing and accumulating β in a culture, and collecting this. 2. The bovine interleukin-1 according to claim 1, wherein the amino acid sequence of the protein having bovine interleukin-1β activity is that of the mature bovine interleukin-1β shown in SEQ ID NO: 1. 1β manufacturing method. 3. The method according to claim 1 or 2, wherein the base sequence of the DNA encoding the amino acid sequence of the protein having bovine interleukin-1β activity is represented by the base sequence of SEQ ID NO: 6. The method for producing bovine interleukin-1β.