JPH11266868A - Transformant, and production of canine interferon-gamma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a transformant useful for producing a large amount of canine interferon-γ used for antitumor agents and antiviral agents for dogs by transforming a yeast cell with a recombinant vector combined with a DNA encoding the canine interferon-γ protein. SOLUTION: This transformant is obtained by ligating a DNA encoding canine interferon-γ protein to a vector expressing in a yeast cell, isolating a plasmid for producing the canine interferon-γfrom the ligation product, and subsequently introducing the plasmid into a yeast cell such as Saccharomyces cerevisiae. The transformant is proliferated, and the produced canine interferon-γ is recovered.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a canine interferon-γ wherein the temporary structure of the protein is derived from canine genetic information.
Transformant obtained by transforming yeast cells, which is intended to be mass-produced by genetic engineering techniques and thereby used as a pharmaceutical product (anti-tumor, anti-virus, anti-allergic), that is, a recombinant yeast, and a method using the same The present invention relates to a method for producing canine interferon-γ.

[0002]

2. Description of the Related Art Interferon is a physiologically active substance mainly composed of a protein which exhibits antiviral activity.
Is abbreviated.

[0003] Advances in genetic engineering technology have made it possible to mass-produce not only human INFs but also animals such as cattle, horses and cats, and as a result, research and development of the use of INFs as therapeutic agents for viral diseases and tumors. Some have been done. As for dogs, α, β, and γ types of INF have been reported. [Adolf et al .: J. Interferon Reserch, 7,173-1
83 (1987) and Devos et al .: J. Interferon Reserch, 12 95
-102 (1992)] As an example of production using yeast, there is an example in which the gene for human hinterferon-γ is incorporated into yeast and expressed (Derynck et al .: Nucledic Acids Research, 11,
1819-1837 (1983)), but no production example of canine interferon-γ is known.

[0004] In dogs, a number of tumors such as mammary gland tumors, a number of viral diseases such as parvovirus infection and distembar infection, and a number of allergic dermatitis are known.

[0005] Therefore, if canine INF is mass-produced and readily available, it is expected that its use as an antitumor, antiviral and antiallergic agent for dogs will be opened.

[0006] Recombinant silkworm nuclear polynucleosis disease virus, which already incorporates DNA encoding the protein of canine interferon-γ, and canine interferon-γ by E. coli.
Methods for producing γ are known (Okano et al .: JP-A-9-2340).
85).

[0007] However, the method using the silkworm nuclear polynucleotic disease virus requires the use of silkworm established cells or silkworm living organisms, which requires an industrially complicated process. Although the method using Escherichia coli is industrially simple,
There is a problem that sugar chains in canine interferon-γ protein do not bind, which may affect biological activity.

[0008]

In contrast to Escherichia coli, protein production using yeast can produce a protein with a sugar chain attached thereto, and can be compared with production methods using silkworm established cells or silkworm organisms. However, a conventional culture apparatus can be used, which is simple. Accordingly, an object of the present invention is to provide a method for mass-producing canine interferon-γ that has solved the above-mentioned disadvantages of the conventional example by this method.
And to provide a transformant obtained by transforming a yeast cell for this method.

[0009]

Means for Solving the Problems In view of such circumstances, the present inventors have aimed at mass production of canine interferon-γ, and have devised ingenuity to express DNA encoding canine interferon-γ protein in yeast cells. A method for producing a canine interferon-γ by isolating a plasmid that produces canine interferon-γ by linking it to a vector and successfully introducing the plasmid into yeast cells to produce canine interferon-γ. And completed the present invention.

That is, the above object of the present invention has been industrially advantageously achieved by the present invention having the following constitution.

[1] A transformant obtained by transforming a yeast cell with a recombinant vector incorporating a DNA encoding a canine interferon-γ protein.

[2] The yeast cell is Saccharomyces cerevisi
The transformant according to the above [1], which is ae.

[3] A method for producing canine interferon-γ, which comprises growing the transformant according to [1] or [2].

[4] Glyoxalase I promoter DN
Encodes A and canine interferon-γ proteins
A recombinant vector incorporating DNA.

[5] A transformant obtained by transforming a yeast cell with the recombinant vector according to [4].

[6] The yeast cell is Saccharomyces cerevisi
The transformant according to the above [5], which is ae.

[7] A method for producing canine interferon-γ, which comprises growing the transformant according to [5] or [6].

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The DNA encoding the canine interferon-γ protein can be obtained, for example, according to a known method (the method described in JP-A-9-234085). That is,
Poly (A) RNA is extracted from canine cells, converted to cDNA, and subjected to the polymerase chain reaction (hereinafter abbreviated as PCR) using primers based on the gene sequence encoding canine interferon-γ. A gene encoding canine interferon-γ can be obtained.

This DNA is ligated to an expression vector that can be expressed in both yeast and Escherichia coli, the plasmid containing canine interferon-γ is increased in Escherichia coli, isolated, then incorporated into yeast cells, and a yeast recombinant is obtained. be able to. Methods for incorporating a plasmid into yeast include a lithium acetic acid method, an electroporation method,
An ordinary method such as a protoplast method can be used.

The construction of this plasmid is such that it has an antibiotic resistance gene such as ampicillin resistance expressed in Escherichia coli as a marker and UR as a marker that works in yeast.
Those having a gene that complements the nucleic acid requirement, such as the A3 gene, are preferred.

The gene for expression of canine interferon-γ has a commonly used promoter and terminator. As the promoter, commonly used promoters such as Gal1-P (galactokinase promoter) and Glo1-P (glyoxalase I promoter) can be used. As the terminator, a commonly used AOD-T (alcohol oxidase terminator) or the like can be used.

The yeast used as a host may be any yeast as long as it can be genetically modified. For example, yeasts well known as host vectors such as Saccharomyces cerevisiae and Candida utilis are used.

The culture method for growing the yeast into which the canine interferon-γ gene has been incorporated may be a general method.
Sugars such as glucose and saccharose, or alcohols such as ethanol and methanol as carbon sources, inorganic nitrogen sources such as ammonium sulfate, organic nitrogen sources such as polypeptone, other inorganic salts, trace metals, vitamins, and yeast extract which is a natural component Use a medium containing

In order to express the canine interferon-γ gene, a substance that induces the expression of a connected promoter is used. For example, in the case of Gal1-P, galactose is contained in a medium, and in the case of, for example, Glo1-P, Use a method of increasing the osmotic pressure of the medium with salt or the like.

The method of recovering canine interferon-γ includes a method of culturing a yeast into which canine interferon-γ has been incorporated, collecting the cells, and crushing the yeast with glass beads.
A general method of disrupting yeast cells, such as a method of disrupting with a French press, is used to recover the contents of the cells.

[0027]

EXAMPLES The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Canine Interferon-γ Gene 5′GTGTTCTTGCGGCCGCAAAATGCAGGCCATGTTTTT on the basis of the N-terminal and C-terminal nucleotide sequences of canine interferon γ
TAAAG 3 '(SEQ ID NO: 1) and 5' CTAGATATGCGGCCGCCTATT
At the two ends of ATTTCGATGCTCTGCG 3 '(SEQ ID NO: 2)
The primer to which the NotI cleavage site was added was synthesized using a DNA synthesizer. Using the recombinant plasmid pETγ containing the canine interferon-γ gene as a template, PCR was performed under the following conditions.
To perform yeast expression vector insertion for canine interferon
-γ gene was obtained.

The PCR conditions are as follows. Template DNAμ
g, 20 pmol of each primer, 20 mM Tris-HCl buffer (p
H8.0), 1.5 mM MgCl2, 25 mM KCl, 100 μg
/ ml gelatin, 50 μM each dNTP, 3 units Amplitaq DNA polymerase (manufactured by PerkinElmer), and add each paper medicine to make a total volume of 100 μl. DNA denaturation conditions at 60 ° C,
The reaction was performed for 25 cycles using a DNA thermal cycler manufactured by Perkin Elmer under the conditions of primer annealing at 72 ° C. for 1 minute and 30 seconds for 30 seconds. This one
% Agarose gel, and a DNA fragment of about 440 bp was prepared according to a conventional method.

Example 2 Preparation of Expression Vector for Yeast 5 ′ GTTTTTGAATTCGCGAAAAGCT based on the base sequence of Glo1-P
TTTTC 3 '(SEQ ID NO: 3) and 5' CATTGTGCGGCCGCAGCTGGT
GGCGTATCT 3 '(SEQ ID NO: 4)
Synthesized with an NA synthesizer. Using the gene containing Glo1-P as a template, PCR was performed under the following conditions to prepare a promoter portion to be incorporated into the expression vector. The PCR conditions are as follows. Μg of template DNA, 20 pmol of each primer, 20
mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl2,
25 mM KCl, 100 μg / ml gelatin, 50 μM each dNTP,
Each paper medicine is added to make 3 units Amplitaq DNA polymerase (manufactured by PerkinElmer), and the total volume is made 100 μl. The DNA denaturation conditions were 60 ° C. for 30 seconds, the primer annealing conditions were 72 ° C. for 1 minute and 30 seconds, using a DNA thermal cycler manufactured by Perkin Elmer Inc. for 25 minutes.
A cycle reaction was performed.

On the other hand, the plasmid pNOTeI shown in the figure was digested with restriction enzymes EcoRI and NotI to cut out unnecessary portions, and the above-prepared Glo1-P was similarly digested with restriction enzymes EcoRI and NotI, and added to the EcoRI and NotI portions of the vector. DNA Ligation Kit
Using Ver.2, the reaction was carried out at 16 ° C. for 30 minutes and ligated. The nucleotide sequences of Glo1-P and the canine interferon-γ gene directly linked to Glo1-P are as shown in SEQ ID NO: 5.

Example 3 Preparation of Yeast Expression Plasmid Containing DNA Encoding Canine Interferon-γ 20 μg each of the DNA fragment obtained in Example 1 and 1 μg of the yeast expression vector obtained in Example 2 Restriction enzyme E
After digestion with coRI and NotI at 37 ° C for 5 hours, the DNA fragment was purified and ligated according to a conventional method. In accordance with a conventional method, E. coli JM105
Was transformed. L containing 100 μg / ml ampicillin
A product containing a gene encoding canine interferon-γ was obtained from colonies growing on B agar medium according to a conventional method. This recombinant plasmid was designated as pGIAγ. FIG.
Shows a restriction map of pGIAγ.

Example 4 Integration of Recombinant Plasmid into Yeast Saccharomyces cerevisiae CBO18 (MATα ura3-52 lys2
-801 ade2-101 trp1- △ 63 his3- △ 200 leu2- △ 1) to YPD
Medium (Glucose 2%, Polypeptone 2%, Yeast extract 1%) 5
One platinum loop was inoculated into a test tube containing mL and cultured at 28 ° C. for 2 days. This preculture solution was inoculated into a 500 mL Sakaguchi flask containing 50 mL of YPD medium so that the OD550 was about 0.1. 15 to 2
After culturing for 0 hour, the cells were collected when the OD550 reached about 1.0. 0.1M Liacetate10mL after washing with 0.1MLiacetate10mL
Was added and centrifuged at 1000 g for 5 minutes while keeping the temperature at 30 ° C. for 1 hour, and 0.5 mL of 0.1 M Liacetate was added to the obtained cells to obtain competent cells. Put 50 μL of competent cells into a 1.5 mL tube, and put 3 μg of the DNA obtained in Example 3 therein.
Incubated at 10 ° C for 10 minutes. Next, add 0.5 mL of 40% PEG4000 (10 mM Tris-HCl buffer (pH 7.5), incubate at 30 ° C for 10 minutes, incubate at 42 ° C for 5 minutes, add 0.9 mL of water, centrifuge for 5 seconds, and perform appropriate Suspend with water and add the minimum agar medium (Glucose 1%, ammonium sulfate 0.5%, KH ₂ PO
_{4 0.1%, MgSO 4 · 7H} 2 O 0.05%, CaCl 2 · 2H 2 O 0.01%, NaCl
0.01%, L-tryptophan L-lysine, L-histidine, adenine each 0.002%, agar 2%) and spread for 3 to 4 days 2
The culture was allowed to stand at 8 ° C. The appeared colonies were purified on a minimal agar medium and then used as recombinants.

Example 5 Culture of Recombinant The recombinant was inoculated into a test tube medium containing 5 mL of a minimum liquid medium and cultured at 28 ° C. for 2 days. The cells were inoculated into an Erlenmeyer flask containing 100 mL of YPD medium, and cultured so that the OD550 became about 1.0. At that time, Na is adjusted to a final concentration of 1.0M.
Cl was added, and the cells were further cultured for 1 hour. Cells were obtained from the culture by centrifugation. The cells were suspended in 20 mM phosphate buffer at pH 7.0, glass beads having a diameter of 0.5 mm were added, and the cells were stirred with a vortex mixer for 3 minutes to break the cells. The supernatant obtained by centrifugation was used as an extract.

Example 6 Activity Measurement The activity of canine interferon-γ was measured as an antiviral activity by the method described in [Prober et al., Science 238, 336-341 (1987)], ie, the CPE method. Vesicular Stomatitis Virus was used as a measuring virus, and dog MDCK (ATCC CCL-34) was used as a susceptible cell.
Was used for the cells. That is, a diluted solution of a sample containing canine IFN-γ was added to canine MDCK cells cultured at 37 ° C. until the cells became confluent on a 96-well microplate, and further cultured at 37 ° C. for 20 to 24 hours. Was induced. Add VSV and add 1
After culturing for 6 to 20 hours, the surviving dog MDCK cells adhering to the microplate were stained with a crystal violet staining solution containing 20% formalin. The amount of crystal violet on the microplate is 570 n
By measuring the absorbance at 50 m.
% Of the canine IFN-γ at the time of survival,
Defined as a unit (1 LU).

The antiviral activity data obtained by this method is 3.4 × 10 ⁶ U / L, and the standard deviation is 32%. The activity of canine interferon-γ in the recombinant yeast cells was found to be at least 10 ⁶ dilution units / L of the culture solution.

[0037]

According to the present invention, a canine antiviral agent,
Canine interferon-γ expected as an antitumor agent and an antiallergic agent can be mass-produced.

[0038]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 41 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GTGTTCTTGC GGCCGCAAAA TGCAGGCCAT GTTTTTTAAA G41 SEQ ID NO: 2 Length: 37 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence: CTAGATATGC GGCCGCCTAT TATTTCGATG CTCTGCG 37 SEQ ID NO: 3 Sequence length: 27 Sequence type : Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GTTTTTGAAT TCGCGAAAAG CTTTTTC 27 SEQ ID NO: 4 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single Chain Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CATTGTGCGG CCGCAGCTGG TGGCGTATCT 30 SEQ ID NO: 5 Sequence length: 1333 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear sequence Type: other nucleic acid DNA sequence: CGAAAAGCTT TTTCAATGGG GATCTTTCTG GTTCGGGACA 40 GTTAAATATA GCATTAACCA GCGGGTTCAA CAGCGTTGGT 80 AAATGGTAAA CTGACATTGT CAACCCTAGC TAAGCCATAG 120 CTTTTCCGTT GAGGAATTTA CAATAAGGTG GTTCCTTTAG 160 TTATAAATTG CAACTGCCAA AATTTTCGGG TAGCGGCAAA 200 ATTAAACGAA AACCTTCTGT CATATTAAAC ATAGTTTAGT 240 GACGCAATGC ACACACACTC ATATATATGT ATATATTTAT 280 CTATATCATA GGTGTAAGGC AAAGTTATCA CGTGAATATA 320 CTGTTTAGTA TACAAGTACG TTGATTTAAC GGCTGAGAGA 360 TTCTCAAACA AAGTAACGCT TAGTTTAGCT AAAGTAGAAT 400 CGAAGCGCAG TGAAGGCAAG ACGAGTTATC CCTTTATCAC 440 AAAATATAAA AAATAGGTAA AGAGGGGGGT GGGGGTGGTT 480 CGGACTTTGC ATCTCGGTGC ATTAGTACCA CTCAGCTTCA 520 GTTATAGGAA TACTGACAAT GTTCTTGAGC CAGGGAGAAC 560 TGAAATTTCC ATACCCTTTA ACCCTTTATT CTTGAGCATA 600 TGATCTTATT TTTCCACTCA GCACAAAATA CACATCCCGC 640 ATATGATCTG GACACTGAAT AAACAAGGGG CTTTACGATG 680 GAGTAGTAGA CCTGGATACG TAGTCACCAC AGGGATCCTA 720 AACTGCGTCA TAGTAAGT TT CTTTGATACT AGAATGTCCC 760 TCTCTCGTAG TAGTTGATAT CCCGTATTTC TAGTTAGTGA 800 CGTTTATAAA TAGGGAGAAA AAAAATCGGA ATAACATTTC 840 CATGCCTTTT TTTTGACACT TCAGACCAGA TACGCCACCA 880 GCTGCGGCCG CAAAATGTCA GGCCATGTTT TTTAAAGAAA 920 TAGAAAACCT AAAGGAATAT TTTAATGCAA GTAATCCAGA 960 TGTATCGGAC GGTGGGTCTC TTTTCGTAGA TATTTTGAAG 1000 AAATGGAGAG AGGAGAGTGA CAAAACAATC ATTCAGAGCC 1040 AAATTGTCTC TTTCTACTTG AAACTGTTTG ACAACTTTAA 1080 AGATAACCAG ATCATTCAAA GGAGCATGGA TACCATCAAG 1120 GAAGACATGC TTGGCAAGTT CTTAAATAGC AGCACCAGTA 1160 AGAGGGAGGA CTTCCTTAAG CTGATTCAAA TTCCTGTGAA 1200 CGATCTGCAG GTCCAGCGCA AGGCGATAAA TGAACTCATC 1240 AAAGTGATGA ATGATCTCTC ACCAAGATCC AACCTAAGGA 1300 AGCGGAATGAGAGAGACGATGATCGATCGATCGATCGATCGATCGATCAGTCGATCAGTCGATCGATCGATCGATCGATCGATCGATCAGGAGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCAGTCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCAG at

[Brief description of the drawings]

FIG. 1 shows a restriction map of pGIAγ.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/21 ABF A61K 37/66 ABF ABH ABHB ADU ADU ADY ADY (C12N 1/19 C12R 1: 865) (C12P 21/02 C12R 1: 865) (72) Inventor Makoto Shirai 1 Toray at 9 Oe-cho, Minato-ku, Nagoya City, Aichi Prefecture Inside the Nagoya Office (72) Inventor Yuichiro Nakaoki 1 Toray at 9 Oe-cho, Minato-ku, Nagoya City, Aichi Prefecture Inside the Nagoya Plant Co., Ltd. (72) Inventor Kenichi Ogawa 9 Toe, 9 Oecho, Minato-ku, Nagoya City, Aichi Prefecture Inside the Nagoya Plant Co., Ltd. (72) Inventor Katsunari Yamada 1 Toray, 9 Oecho, Minato-ku, Nagoya City, Aichi Prefecture Nagoya Works Co., Ltd.

Claims (7)

[Claims] 1. A transformant obtained by transforming a yeast cell with a recombinant vector having a DNA encoding a canine interferon-γ protein incorporated therein. 2. The transformant according to claim 1, wherein the yeast cell is Saccharomyces cerevisiae. 3. A method for producing canine interferon-γ, comprising growing the transformant according to claim 1 or 2. 4. A recombinant vector comprising a glyoxalase I promoter DNA and a DNA encoding a canine interferon-γ protein. 5. A transformant obtained by transforming a yeast cell with the recombinant vector according to claim 4. 6. The transformant according to claim 5, wherein the yeast cell is Saccharomyces cerevisiae. 7. A method for producing canine interferon-γ, which comprises growing the transformant according to claim 5 or 6.