JPH0892292A - Protein having biological activity of canine interferon-β - Google Patents

Abstract

(57) Abstract: A large amount of canine interferon-β is produced by using a microorganism transformed with a plasmid capable of producing a protein having a biological activity of canine interferon-β. SOLUTION: A gene fragment encoding a protein having an amino acid sequence having a biological activity of canine interferon-β is integrated, and Escherichia coli transformed with a synthetic plasmid capable of producing this protein is cultured and produced. The above-mentioned protein is isolated and collected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to canine interferon-β (hereinafter sometimes abbreviated as “CaIFN-β”) whose primary structure of protein is derived from canine genetic information.
And a protein having the biological activity of CaIFN-β
The present invention relates to a gene fragment encoding a protein having biological activity, a synthetic plasmid produced for the purpose of producing a pharmaceutical product by mass-production of the protein by a genetic engineering technique, and a transformed microorganism thereof.

[0002]

BACKGROUND OF THE INVENTION Interferon (hereinafter sometimes abbreviated as "IFN") is a protein secreted by eukaryotic cells after viral infection or other stimuli, and acts on the cells to cause resistance to the virus. It is known as a substance that induces sex or has a regulatory action in immune cells and further affects cell differentiation and growth of tumors and the like.

At present, five types of IFN are known, and IFN-α, IFN-β, IFN-γ, IFN-ω,
Called IFN-τ, they differ in structure and action.

Due to advances in gene manipulation techniques, not only human IFN but also several mammalian IFNs have been cloned. Among these, IFN-α is bovine (Va
lanet al, 1985 J.Biol.Chem. 260: 5498-5504), horse (Himmler et al, 1987 J. Interferon Res. 7: 173-183).
), Cat (Nakamura et al. 1992, Bisoci.Biotech. Bi
ochem. 56: 211-214), IFN-β is
The gene has been isolated in eung et al, 1984 Bio / Technology 2: 458-464) and horse (Himmler et al, 1986 DNA 5: 345-356), enabling mass production. as a result,
In some cases, researches have been conducted on the use of IFN as a therapeutic drug for viral diseases and tumors (JP-A-58-2246).
90, JP-A-62-501469, JP-A-63-2
No. 33794, JP-A-2-195884, JP-A-2-
304100).

Also in dogs, α and γ types of IFN
Gene cloning has been reported (Himmler et a
l, 1987 J. Interferon Res. 7: 173-183, Zuckeret al, 19
92 J. Interferon Res. 12: 191-194).

[0006] By the way, although it has been reported that IFN is cloned, it is generally difficult to establish a method for producing IFN in large quantities using animals or cultured cells. Neither cloning nor reported.

However, given the fact that a large number of viral diseases are known to dogs and no effective therapeutic agents other than vaccines are known, if dog IFNs were readily available, then dogs' anti-infectives could be It is expected to be used as a viral agent and an antitumor agent.

[0008]

In view of such a situation, the present inventors have isolated and identified the CaIFN-β gene,
And repeated research for mass production of CaIFN-β,
The present invention has been completed by identifying a transformant capable of identifying CaIFN-β and a gene fragment encoding the CaIFN-β and mass-producing this CaIFN-β.

That is, a first object of the present invention is CaIF.
A protein having a biological activity of N-β, and
It is intended to provide a gene fragment encoding a protein having a biological activity of CaIFN-β.

Another object of the present invention is the above CaIF.
It is an object of the present invention to produce a transformed plasmid capable of producing a protein having an N-β biological activity and to provide a transformed microorganism having this plasmid.

A further object of the present invention is to provide a production method capable of mass-producing CaIFN-β using this transformed microorganism.

[0012]

One of the features of the present invention is:
It is intended to provide a protein having the biological activity of CaIFN-β having the amino acid sequence of SEQ ID NO: 1, and a gene fragment encoding the protein having the amino acid sequence of SEQ ID NO: 1. In the present invention, the protein having the amino acid sequence of SEQ ID NO: 1 is CaIF.
The polypeptide of SEQ ID NO: 1 having N-β biological activity itself or a polypeptide containing a part thereof is meant, and the gene fragment encoding the polypeptide of SEQ ID NO: 1 encodes the polypeptide. The gene fragment itself, or a sequence necessary for expressing this, such as a promoter or terminator, which acts as an autonomous unit for expression of the polypeptide.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION A protein having the biological activity of canine interferon-β (CaIFN-β) having the amino acid sequence of SEQ ID NO: 1 was identified as follows. That is, DNA is extracted from the liver of a dog and the target gene region is amplified by the PCR (polymerase chain reaction) method using a synthetic DNA primer prepared based on a gene sequence already isolated from other mammals. Incorporating this into a commercially available vector, determining the nucleotide sequence, selecting a clone having homology to the IFN-β gene of another species, and using this to transform commercially available E. coli to express in E. coli. , A plasmid capable of producing a protein having antiviral activity was selected.
The plasmid having the above activity is called p31-1, and E. coli transformed with this plasmid is called 31-1.

The above operation is performed by Sam Block et al.
rook et al, 1989 Molecular cloning: a laboratory m
anual.2nd edition cold spring harbor, New York: co
ldspring harbor press) and other known gene manipulation technologies
It can be performed according to cell engineering techniques. In addition, the above-mentioned antiviral activity was measured using cultured dog cells and VSV (Vesicu).
lar stomatitis virus) and Armstrong (Armstrong, 1981 Meth.Enzymol. 78: 381-387) CP
Method E can be used.

The protein having the biological activity of CaIFN-β having the amino acid sequence of SEQ ID NO: 1 of the present invention is the one transcribed / translated from the nucleotide sequence of SEQ ID NO: 1 or the genome encoding CaIFN-β. It is not always necessary and may be produced by, for example, a chemical synthesis method or expression of a transformant.

The base sequence encoding the amino acid of SEQ ID NO: 1 may be obtained by any method such as chemical synthesis method, DNA replication method, reverse transcription method and transcription method.

Another feature of the present invention is that a gene fragment encoding a protein having the biological activity of CaIFN-β having the amino acid sequence of the above SEQ ID NO: 1 is incorporated, and this protein can be produced. Synthetic plasmids, such plasmids are for example p31 above.
Offered as -1.

Still another feature of the present invention is that a gene fragment encoding a protein having the amino acid sequence of SEQ ID NO: 1 is incorporated and a host cell is transformed with a synthetic plasmid capable of producing this protein. The purpose is to provide transformed microorganisms.

Such CaIFN-β producing cells can be produced as follows. When the host cell is a eukaryotic cell, for example, CaIF extracted from 31-1 above
CaIFN-β producing cells can be produced by binding a gene fragment encoding a protein having N-β biological activity to a promoter such as a virus and transforming eukaryotic cells. When a recombinant viral vector such as baculovirus is used, the recombinant viral vector can be produced, for example, based on the method of Smith et al. (Mol. Cell. Biol. 3: 2156-2165). Ie C
A gene fragment encoding a protein having aIFN-β biological activity is ligated to a multinucleate promoter of a transfer vector of baculovirus, and the transfer vector is introduced into baculovirus DNA and insect cells,
Of the obtained recombinant baculovirus, CaIFN-
A recombinant clone producing β activity is selected, and the recombinant clone is infected with insect cells or insects, and CaI
FN-β protein can be produced.

When the host cell is a prokaryotic cell, for example, a gene fragment encoding a protein having a biological activity of CaIFN-β is ligated to an expression vector and the prokaryotic cell is transformed to produce a CaIFN-β producing cell. can do.

[0021] The most preferable one of such microorganisms is Escherichia coli, and the applicant of the present invention has established a typical strain 31-1 as of July 18, 1994 by the Ministry of International Trade and Industry Deposited at the Institute of Engineering and Technology,
The accession number of (FERM P-14432) is given.

Still another feature of the present invention is a method for producing the above-mentioned transformed microorganism by culturing the protein and separating and recovering the produced protein having the biological activity of CaIFN-β.

Although the above-mentioned protein can be separated from the cultured cells by a usual method, a method by affinity chromatography, particularly a method using a carrier to which a specific antibody is bound is preferably used.

According to the present invention, a large amount can be produced using a microorganism or the like transformed with a protein having the biological activity of CaIFN-β.

[0025]

EXAMPLES The present invention will be described in more detail below with reference to examples.

(1) By PCR of CaIFN-β gene
Amplified 5-week-old dog liver was excised, cut into small pieces with surgical scissors, and TE (10 mM Tris-hydrochloric acid (pH 8.0) -1 containing 0.6% SDS (sodium dodecyl sulfate) -1.
The protein was suspended in 100 ml of mM EDTA (ethylenediamine tetraacetate), proteinase K was added to 100 μg / ml, and the protein was digested overnight at 37 ° C. with stirring with a stirrer. The mixture is extracted with phenol / chloroform, the aqueous layer is removed, and 1
After adding 0 ml of 3M sodium acetate (pH 5.2), 200 ml of ethanol was added and the mixture was cooled at -20 ° C for 2 hours. Centrifuge to remove 10 pellets from the bottom.
It was dissolved in ml of sterile distilled water. In addition, based on the known human, mouse, horse, and bovine base sequences, D having the following base sequence in a portion relatively conserved among these animal species
The NA primers, COM and CIB7, were synthesized.

COM 5 ′-TGAAAGTGGGAAATTCCTCT CIB7 5 ′-TGTCCTTTCATATGCAGT and using these primers, dog genomic DNA
1 μg, 3 μM of each primer, 20 each
0 μM deoxynucleotide, 10 mM KCl, 1
1 mM to 0 mM Tris-HCl, 2 mM magnesium chloride
0 units of commercial Vent polymerase (New Engl
and Biolab. Inc.), and paraffin oil is overlaid, and the temperature is 94 ° C. for 45 seconds, 50 ° C. for 60 seconds, and 72 ° C. for 12 seconds.
PCR was performed 30 cycles with 0 second as one cycle.

Removing paraffin oil from the product,
The amplified DNA fragment was analyzed by electrophoresis. That is, by performing agarose gel electrophoresis, a DNA fragment portion of about 900 bases was cut out, and this DNA fragment was blunt-ended with Klenow enzyme and phosphorylated, and then commercially available pG
A recombinant plasmid was obtained by inserting it into the SmaI site of the EM3Z vector (Promega Co.).

Next, the reaction solution containing this recombinant plasmid was mixed with competent Escherichia coli JM109, and a transformation reaction was carried out to obtain a transformant CaIB. Next 100μ
The clones grown on the LB plate containing g / ml ampicillin were LB containing 100 μg / ml ampicillin.
Incubate in 2 ml of medium (37 ° C, overnight), extract the plasmid by the alkaline miniscreen method, and use EcoRI and Hin.
A clone was selected which had been cut with dIII and incorporated a DNA fragment of the desired size.

Of these, 12 clones were selected and the nucleotide sequence was determined as follows.

(2) Determination of nucleotide sequence Recombinant plasmids were extracted from the 12 clones obtained above,
After purification, the reaction was carried out by the dideoxy sequencing method using a commercially available cycle sequence kit (manufactured by Perkin Elmer Japan) according to the attached protocol, and the nucleotide sequence was determined using a sequencer of Perkin Elmer Japan.

That is, first, the nucleotide sequences at both ends of the inserted DNA fragment were determined using a sequence primer complementary to the pGEM3Z vector portion. Then
From these 12 clones, 6 clones having homology with the human IFN-β gene were selected and further inserted into the DNA.
Synthesize the sequence primer toward the inside of the fragment,
The base sequence was determined, and by repeating such operations, the base sequence of the DNA shown in SEQ ID NO: 1 was finally determined. 6
All the clones retained a DNA fragment having the same nucleotide sequence.

The base sequence data thus obtained was compared with the base sequences of four other known IFN-β,
The results are shown in FIGS. 2 to 5 and FIG. 6, and the matching rates are summarized in Table 1 below. 2 to 5 show human, mouse,
FIG. 6 shows the results in which the nucleotide sequences of the equine and bovine IFN-β genes and the CaIFN-β gene are aligned so that the homologous portion is maximized.
The results are shown in which the amino acid sequences predicted from the nucleotide sequences of the β gene and CaIFN-β gene are arranged side by side so that the homologous portion is maximized. In these figures, “.” indicates CaIF
In the case of the same nucleotide sequence as N-β, “−” indicates the case where a deletion is recognized. There are three or two IFN-β genes in bovine and horse, respectively, and the β1 gene was used as a representative for analysis.

[0034]

[Table 1]

As can be seen from this result, the obtained DN
The base sequence of the A fragment is 4 kinds of IFN-
About 70% homology with the nucleotide sequence of β gene was observed. The predicted amino acid sequence is also 40-60.
% Homology.

From the results of the amino acid sequences shown above, it was revealed that the CaIFN-β gene has a signal sequence at the N-terminal which is 11 amino acids longer than that of IFN-β of other species. In addition, the positions of cysteine residues, which are said to be influenced by the secondary structure and the tertiary structure, were conserved at all three positions. Furthermore, the signal peptide that is cleaved when secreted was presumed to be from the 1st amino acid to the 31st amino acid, and the mature molecule was presumed to have the 32nd methionine at the N-terminal.

Whether or not the above-mentioned DNA fragment-derived protein has antiviral activity was examined using the protein produced by Escherichia coli produced in (3) below.

(3) Production of Protein-Producing Bacteria FIG. 1 shows a method for producing an expression plasmid. That is,
The following DNA primers BF3 and BR3 so that the BamHI and EcoRI parts of the pGEX-2T vector (manufactured by Pharmacia) to be expressed as a glutathione-S-transferase fusion protein have the same amino acid frame as the glutathione-S-transferase protein. Was synthesized.

BF3 5'-GCCGGGATCCATGAGCAACGACTTGCTTCG-3 'BR3 5'-GGCCGAATTCTCAGTTCTGGAGATAATCTG-3' The desired DNA fragment was obtained by PCR from one of the above plasmids (p31), and this DNA fragment was labeled with B
Cleaved with amHI and EcoRI, and similarly cut with BamHI and E
The pGEX-2T vector cleaved with coRI was ligated with T4-ligase to obtain a plasmid p31-1.

Escherichia coli J obtained by making this reaction solution competent
A transformant was obtained by mixing with M109 and carrying out a transformation reaction. Then L containing 100 μg / ml ampicillin
The clones growing on the B plate were cultured in 2 ml of LB medium containing 100 μg / ml of ampicillin (37 ° C., overnight), the plasmid was extracted by the alkaline miniscreen method, and cut with EcoRI and BamHI to obtain the desired size. The clone 31-1 incorporating the DNA fragment was selected.

(4) Protein as fusion protein
Production of GS using the above-mentioned Escherichia coli 31-1 which is a transformant
Using T purification modul (Pharmacia),
The fusion protein was produced based on the attached manual.

That is, culture was carried out for 1.5 hours from the initial concentration OD600 = 0.1, and IP was adjusted to a concentration of 0.1 mM.
TG (isopropyl β-D-thiogalactoside) was added, and the mixture was further incubated at 28 ° C. for 4 hours to produce a fusion protein in E. coli. E. coli is collected by centrifugation and PBS
After being suspended in (-), the bacteria were destroyed by ultrasonic treatment. The fusion protein was solubilized with 1% sodium sarcosinate and then loaded onto a glutathione sepharose column for binding. The fusion protein was eluted with Tris buffer containing 10 mM glutathione. Thrombin was added to 10 units / mg protein and left at room temperature for 18 hours to allow glutathione-S-transferase protein and CaIF.
The N-β protein was cleaved off. After removing glutathione by dialysis, glutathione-S-transferase and CaIFN
The -β mixture was applied to a glutathione column, and the flow-through fraction was used as a CaIFN-β sample.

(5) Method for measuring antiviral activity The CaIFN-β sample activity described above was measured using the above-mentioned Armstrong (Armstrong, 1981 Meth. Enzymol. 78: 381-387).
MDC as follows with reference to the method described in
It was measured by the results of CPE inhibition using K cells and VSV (Vesicular stomatitis virus).

That is, 0.5% fetal bovine embryos (FB) were prepared so that the number of dog-derived cultured cells MDCK was 10 ⁵ cells / ml.
Suspended in Dulbecco's modified Eagle's minimum essential medium (abbreviated as DMEM) containing S), and 0.1 ml each was dispensed into the wells of a 96-well multi-well plate. After culturing for about 1 day, discard the culture broth and add DMEM containing 0.5% FBS.
The CaIFN-β sample diluted with
It was made to act on DCK cells. Discard the culture medium and the sample, and
Infecting VSV in the presence of DMEM containing 5% FBS,
After 2 days, cytopathic change caused by VSV-infected growth was observed, and the degree of damage to the entire sheet of MDCK cells was quantified and graphed. From the graph, the reciprocal of the dilution rate of the specimen expected to show 50% cytopathic activity is shown as IFN activity.

According to this, the IFN activity of the purified CaIFN-β sample was measured to be 10 ⁸ units per mg, which was similar to that of human and mouse IFN-β. Therefore, it was confirmed that the protein of SEQ ID NO: 1 is a protein having the biological activity of CaIFN-β.

(6) Preparation of Baculovirus Transfer Vector FIG. 7 illustrates the procedure for preparing a baculovirus transfer vector. That is, the following DNA primer BF-Ba is included so as to include the entire open reading frame containing the signal peptide of the CaIFN-β gene.
c, BR-Bac were synthesized. The 7th to 9th bases of the BF-Bac primer, ATG, correspond to the translation initiation signal of this gene. In addition, the 5th to 10th bases of the BR-Bac primer, GAATTC,
This is a recognition sequence of the restriction enzyme EcoRI added so that the forward direction or the reverse direction can be distinguished from the promoter after inserting the transfer vector.

BF-Bac 5′-GGCCCCATGGTAATAGGTGAGACTCTTCCC-3 ′ BR-Bac 5′-GGCCGAATTCTCAGTTCTGGAGATAATCTG-3 ′ That is, among the plasmids in which the PCR-amplified DNA fragment was first cloned, the dog IFN-β shown in SEQ ID NO: 1 was used.
Choose a gene having the nucleotide sequence of the gene, use it as a template, and use the BF-Bac and BR-Bac primers to perform P
D including all open reading frames by CR method
The NA fragment was amplified.

That is, 0.1 μg / 50 of the present plasmid
μl, 1 μM each primer, 200 μM deoxynucleotide, 10 mM potassium chloride, 10 m
M Tris-HCl (pH 7.5), 2 mM magnesium chloride, and 10 units of commercially available Vent polymerase (New England Biolab. Inc) were added to give a total volume of 50 μl.
And layer with paraffin oil for 45 seconds at 94 ° C for 5
P for 60 seconds at 0 ° C and 120 seconds at 72 ° C as one cycle
20 cycles of CR were performed. Paraffin oil was removed by adding chloroform, and the amplified DNA fragment was confirmed by agarose electrophoresis.

2 units of Klenow enzyme (manufactured by Takara Shuzo) was added to the reaction solution, and the mixture was allowed to stand at room temperature for 25 minutes to smooth the DNA fragment. After the Klenow enzyme was removed by phenol extraction, ethanol precipitation was performed and the DNA sample was concentrated. 1% agarose gel electrophoresis was performed to cut out the target agarose block containing a DNA fragment of about 0.6 kb. The DNA fragment is Gene Clean Kit II (Bio
101, Inc.) was used and purified according to the attached protocol.

The DNA fragment was treated with 10 mM Tris-HCl (pH
8.0) Dissolved in a buffer containing 5 mM magnesium chloride, added ATP to 5 mM, and phosphorylated the 5 ′ end of the DNA fragment with 10 units of polynucleotide kinase (Boehringer Mannheim). .

On the other hand, transfer vector DNA was prepared as follows. Ba of pBacPAK1 transfer vector (CLONOTECH Laboratories, Inc)
The transfer vector pBacPAK-IS in which the SmaI linker was inserted at the mHI site was cleaved with SmaI, and the phosphate group at the 5 ′ end was removed with 2 units of bacterial-derived alkaline phosphatase (BAP, manufactured by Takara Shuzo). The bacterial-derived alkaline phosphatase was inactivated by treatment at 55 ° C. for 30 minutes, the enzyme was removed by phenol extraction, and the vector DNA was concentrated by ethanol precipitation.

PCR-amplified and post-treated 100 fmol of DNA fragment and pBacPAK-IS vector DNA
40 fmol was mixed, a ligation reaction was performed, the reaction solution was mixed with competent Escherichia coli K12-derived TB1, and a transformation reaction was performed to obtain a transformant. Next, a clone that grows on an LB plate containing 100 μg / ml ampicillin was cultured in 2 ml of LB medium containing 100 μg / ml ampicillin at 37 ° C. overnight, a plasmid was extracted by an alkaline miniscreen method, and cut with BamHI, A clone having a plasmid containing an insert of about 0.6 kb was selected and designated as Bac-β series. The plasmids possessed by these transformants were designated as pBac-β series.

Since the inserted DNA fragment was ligated with blunt ends, pBac-β series plasmids were mixed with 1: 1 plasmids having inserts bound to the baculovirus multinucleus promoter in the forward and reverse directions. (Fig. 8). EcoRI and Hin in the forward direction to the polynucleoprotein promoter
A 450 bp band appears when cleaved with dIII, and a 1040 bp band similarly appears in the opposite direction. The resulting plasmid was cleaved with EcoRI and HindIII, and the orientation of the insert of each DNA clone with respect to the promoter was determined.

E. coli clone B harboring plasmid pBac-β3 with inserts forward to the promoter
Select ac-β3, incubate in 100 ml, plasmid D
NA for Qiagen columns (DIAGEN, GmbH, QIAGEN, In
(manufactured by Company c) according to the attached protocol.

(7) Preparation of Recombinant Baculovirus FIG. 9 shows an outline of the method for preparing recombinant baculovirus.
Transfer vector DNA and restriction enzyme Bsu36I
When the viral DNA cleaved in step 2 was adsorbed to lipofectin and added to Sf21AE cells, the DNA-lipofectin complex was taken up by Sf21AE cells and, although at low frequency, homologous recombination occurred between the two DNAs, resulting in the combination in the transfer vector. The replacement gene is baculovirus DN
Bsu inserted into A and at the same time the restriction enzyme Bsu of viral DNA
The break point due to 36I is removed. Cleaved viral DNA that has not undergone homologous recombination is unable to repair the breakpoint and rarely propagates.

Therefore, since only the recombinant viral DNA replicates and proliferates to form virus particles and clear plaques are formed on the cell sheet, the recombinant virus can be isolated using the plaques as an index. This method is generally widely used.

A specific method will be shown below.

Sf21AE cells were cultured in Grace's medium (manufactured by Gibco) containing 10% immobilized fetal bovine serum (FBS) and 0.26% bactotryptose broth (BTB), and 1 × 10 ⁶ cells / well were prepared. The mixture was dispensed into a 6-well plate and incubated at room temperature for 1 hour or more to adhere cells to the bottom surface of the plate well.

On the other hand, 1 μg of pBac-β3 transfer vector DNA and B digested with the restriction enzyme Bsu36I
acPAK6 viral DNA (CLONOTECH Laboratorie
20 ng (manufactured by S. Inc.) was mixed to make a total volume of 8 μl. 8 μl of lipofectin (manufactured by BRL) was added, mixed, and allowed to stand at room temperature for about 15 minutes to form a DNA-lipofectin complex.

Sf21AE cells prepared as described above were washed with serum-free Grace's medium and 1 ml Grace's medium was added. 16 μl of DNA-lipofectin complex was added thereto, and the mixture was cultured at 27 ° C. for 5 hours. 1 ml 1
Grace's medium containing 0% FBS and 0.26% BTB was added, and the mixture was further cultured at 27 ° C for 3 days, and formation of plaques was confirmed. Aseptically remove the culture supernatant, and use a portion of IF
N activity was measured and the rest was used for plaque cloning.

Plaque cloning was performed as follows.

The culture supernatant was serially diluted 10 times, 200 μl of the diluted culture supernatant was added to Sf21AE cells adhered to a 6-well plate, and the mixture was allowed to stand for 1 hour to adsorb the virus to the cells. 2 ml of Grace's medium containing 1% agarose /
Layer in holes and add 1 ml of 10% FBS and 0.26%
Grace's medium containing BTB was overlaid and cultured at 27 ° C. for 3 days. In order to easily observe the plaques that appeared with the naked eye, 1 ml of PBS (-) containing 0.01% neutral red was overlaid and left overnight. Select clear plaques that do not produce polynuclear bodies, cut out viral particles secreted in agarose together with agarose, and add 0.5 ml of 10% FB.
It was put in a Grace's medium containing S and 0.26% BTB, and dispersed by ultrasonic waves. Centrifuge at 3000 rpm for 5 minutes,
The supernatant was used as a recombinant baculovirus sample at 4 ° C.
, And used as material for the next plaque cloning.

This operation was repeated 3 times, and the cloned recombinant baculoviruses IBacβ1 and IBacβ2 were cloned.
Got

(8) CaIF of recombinant baculovirus
N-β production IBacβ1 and IBacβ2 at Sf21 at moi = 10
AE cells were infected, and the culture supernatant was sampled over time to examine the production of IFN activity (Table 2). The IFN activity was measured by the following procedure using A72 cells.

[0065]

[Table 2]

[0066] Cell cultures from canine, A72 L-15 (Dainippon Pharmaceutical Co., Ltd. containing with 20% FBS to be 3 × 10 ⁵ cells / ml and (Dainippon Pharmaceutical Co., purchased from) )
Then, 0.1 ml each was dispensed into a 96-well multiwell plate. After culturing for about 1 day, the culture solution is discarded and 5
A sample diluted with L-15 containing% FBS was added, and the sample was allowed to act on A72 cells for 1 day. Discard the culture solution and the sample, and use 10 mM HEPES (pH 7.4) and 5% FBS.
VS-15 (Vesicular stomatitis in the presence of L-15 containing
24 hours later, the cytopathic effect caused by VSV infection proliferation was observed, and the extent of damage to the entire sheet of A72 cells was quantified and graphed. 5 from the graph
The reciprocal of the dilution rate of the specimen expected to show 0% cytopathicity was shown as IFN activity.

IFN activity was found 24 hours after infection in the culture medium of both recombinant baculovirus-infected cells.
It reaches its maximum after 72 hours and the production volume is about 10 ⁵ units /
IFN activity of ml was reached. IFN of both clones
Active production was about the same. No IFN activity was found in the culture supernatant of uninfected controls.

[0068]

INDUSTRIAL APPLICABILITY According to the present invention, a protein having a biological activity of CaIFN-β can be produced in a large amount using a transformed microorganism, and an effective therapeutic agent other than a vaccine is not known in dogs. It is possible to obtain the effect that antiviral agents, antitumor agents and transfer of this protein can be expected.

[0069]

[SEQ ID NO: 1] 10 20 30 40 50 60 TGGGAAATTCCTCTGCGATAGAAAGAGGACCATCCTGTATAAATAGGGCTACACTCCTGG 70 80 90 100 110 120 TAGAAGGGCATTTGCGCTCTGAACTCTCAGAGCTTTGGCTTCAGCATCGAGATGGTAATA MetValIle 130 140 150 160 170 180 GGTGAGACTCTTCCCATTTTCATCATGACCAGTAGATGCATCCTCCAAACAACTCTCCTG GlyGluThrLeuProIlePheIleMetThrSerArgCysIleLeuGlnThrThrLeuLeu 190 200 210 220 230 240 TTGTATTTCTCCACCATGGCTCTTGCCATGAGCAACGACTTGCTTCGATCCCAGCTAAGC LeuTyrPheSerThrMetAlaLeuAlaMetSerAsnAspLeuLeuArgSerGlnLeuSer 250 260 270 280 290 300 AGCAGCAGTTTGGAGTGTCAGGAGCTCCTATTACAGTTGAATGGAACCACTGAATATTGC SerSerSerLeuGluCysGlnGluLeuLeuLeuGlnLeuAsnGlyThrThrGluThrCys 310 320 330 340 350 360 CTCAAGGACAGGATAAACTTCGAGATCCCTGAGGAAATCGAGAAATCACGCCAGTTCCAG LeuLysAspArgIleAsnPheGluIleProGluGluIleGluLysSerArgGlnPheGln 370 380 390 400 410 420 AAGGAGGACATCATATTGATCACCCATGAGATGTTCCAGAAGATCTTTGATATTTTCAGG LysGluAspIleIleLeuIleThrHisGluMetPheGlnLysIlePheAspIlePheArg 430 440 450 460 470 480 AGAAATATCTCTAGAACAGGATGGAATGAGACCACTGTCGAGAACCTTCTTGT GAAGCTC ArgAsnIleSerArgThrGlyTrpAsnGluThrThrValGluAsnLeuLeuValLysLeu 490 500 510 520 530 540 CACTGGCAGAAGGAACATCTGGAGATAATCCTGGAGGACGTCAAAGAGAAGGAAAACTTC HisTrpGlnLysGluHisLeuGluIleIleLeuGluAspValLysGluLysGluAsnPhe 550 560 570 580 590 600 ACCTGGGACAACAGGACTCTTCTGCACCTGAAGAAATATTACTTAAGGATTGTGCAGTAC ThrTrpAspAsnArgThrLeuLeuHisLeuLysLysTyrTyrLeuArgIleValGlnTyr 610 620 630 640 650 660 CTGAAGGCCAAGGAGTACAGCATCTGTGCCTGGACAATAGTCCAAGCAGAAATCTGCAGG LeuLysAlaLysGluTyrSerIleCysAlaTrpThrIleValGlnAlaGluIleCysArg 670 680 690 700 710 720 AACTTTTTCTTCCTTAATATACTTACAGATTATCTCCAGAACTGAAGATCTCTCAGCCTC AsnPhePhePheLeuAsnIleLeuThrAspTyrLeuGlnAsn *** 730 740 750 760 770 780 TGCCTCTCGGAATGGACGATGCTTGACAAGGATTTCAGGCATTCCTCAACTGGCAGATGC 790 800 810 819 TCTGTAAGTGACAGCTAATGTACTGCATTTGAAAGGACA

[Brief description of drawings]

FIG. 1 shows the construction of a plasmid expressing the CaIFN-β gene in Escherichia coli.

FIG. 2 shows the results in which the nucleotide sequences of human, mouse, horse, bovine IFN-β gene and CaIFN-β gene are arranged side by side so that the homologous portion is maximized. However, CaIFN-
When the nucleotide sequence is the same as that of the β gene, it is indicated by ".", and when deletion is observed, it is indicated by "-".

FIG. 3 shows a continuation of FIG.

FIG. 4 shows a continuation of FIG.

FIG. 5 shows a continuation of FIG.

FIG. 6 Human, mouse, horse, bovine IFN of FIGS.
The figure which showed the result in which the amino acid sequences predicted from the base sequences of the -β gene and the CaIFN-β gene were arranged side by side so that the homologous portion was maximized.

FIG. 7 is a diagram for explaining a method for producing a baculovirus transfer vector.

FIG. 8 is a diagram illustrating a method for examining the direction of a DNA fragment inserted by the method of FIG.

FIG. 9 is a diagram for explaining a method for producing a recombinant baculovirus.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 5/10 7/00 8931-4B 15/09 ZNA C12P 21/02 F 9282-4B // A61K 38/21 ADU ADY (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/02 F C12R 1:19) A61K 37/66 ADU F ADY F (C12N 5/00 B C12R 1:91)

Claims (8)

[Claims] 1. A protein having the biological activity of canine interferon-β having the amino acid sequence of SEQ ID NO: 1 or a part thereof. 2. A gene fragment encoding a protein having the amino acid sequence of claim 1 or a part thereof. 3. A synthetic recombinant viral vector into which a gene fragment encoding a protein having the amino acid sequence of claim 1 or a part thereof is incorporated and which can produce the protein. 4. A synthetic plasmid into which a gene fragment encoding a protein having the amino acid sequence of claim 1 or a part thereof is incorporated to produce this protein. 5. A eukaryotic cell obtained by transforming a host cell with a synthetic plasmid into which a gene fragment encoding a protein having the amino acid sequence of claim 1 or a part thereof is incorporated and which is capable of producing this protein. 6. A prokaryotic cell obtained by transforming a host cell with a synthetic plasmid into which a gene fragment encoding a protein having the amino acid sequence of claim 1 or a part thereof is incorporated and which is capable of producing this protein. 7. The Institute of Biotechnology, Institute of Biotechnology, which has a gene fragment encoding a protein having a part of the amino acid sequence of claim 1 and is transformed with a synthetic plasmid capable of producing this protein. Escherichia coli of deposited FERMP-14432. 8. Produced by infecting insect cells with the recombinant baculovirus that is the viral vector of claim 3, or culturing the transformed cells of claim 5 or 6, or Escherichia coli of claim 7. A method for producing a protein having a biological activity of canine interferon-β, which comprises separating and recovering the protein according to claim 1.