JP2786888B2 - Method for producing fish growth hormone - Google Patents

Description

The present invention relates to a gene encoding a fish growth hormone polypeptide, a recombinant DNA incorporating the gene, and a recombinant yeast containing the recombinant DNA. The present invention also relates to a method for promoting the growth of a wide range of fish by using the yeast growth hormone polypeptide by the yeast.

[Conventional technology]

Fish growth hormone is biosynthesized in the pituitary gland,
Until now, tilapia [SNFarmer, Gen.Comp.Endcr
in., 30, 91 (1976)], and growth hormones have been isolated from various fishes and their properties have been elucidated. Further, tuna (Japanese Patent Application Laid-Open No. 104180), white salmon (Japanese Patent Application Laid-Open
-15699, Japanese Patent Application Nos. 59-213361 and 60-50096), a gene encoding a growth hormone polypeptide was isolated, and E. coli containing recombinant DNA incorporating these genes was cultured. Methods for obtaining growth hormone polypeptides have been reported.

However, it is only possible to obtain a growth hormone polypeptide by culturing a yeast containing a recombinant DNA in the case of chum salmon growth hormone.
Only reported in 4297.

[Problems to be solved by the invention]

Fish growth hormone has a fish growth promoting effect and is a very useful future feed in cultivation fisheries. However, it is impossible at present to supply the hormone inexpensively and in large quantities, because it requires a large amount of pituitary glands of fish.

The present inventors have previously described a recombinant DNA in which a tuna growth hormone polypeptide has incorporated a tuna growth hormone gene.
By cultivating Escherichia coli containing E. coli, it was found that a significant amount of tuna growth hormone polypeptide was produced in the culture. However, endotoxins are often present in protein preparations from E. coli, and the purification steps to remove them are not very economical. Many of the proteins produced by Escherichia coli from higher organisms have many problems such as accumulation as inactive proteins because the biochemical environment in Escherichia coli is different from that of higher organisms. ing. The present inventors have further researched and made intensive studies to develop a method for inexpensively supplying a large amount of fish growth hormone which does not contain endotoxin and has activity, and completed the present invention.

[Means for solving the problem]

The present invention relates to a recombinant D containing a tuna growth hormone-encoding DNA containing the amino acid sequence shown in FIG.
The present invention relates to a method for producing fish growth hormone by producing a large amount of tuna growth hormone in a culture by culturing a yeast containing NA, and collecting the tuna growth hormone from a culture medium, and a feed for seafood containing the same. According to the present invention,
A fish growth hormone having no endotoxin and having activity can be produced.

 Hereinafter, the present invention will be described in detail.

Total RNA was extracted from fresh pituitary gland of tuna and passed through an oligo dT cellulose column, and RNA containing polyadenylic acid (poly A) (poly A · R)
Prepare NA). Next, using this total poly-A RNA as a template,
Synthesize double-stranded DNA with reverse transcriptase. Recombinant DNA
Is prepared by recombinant DNA techniques.

Synthesis of cDNA and incorporation of cDNA into vector
Okayama-Berg method [Okayama &Berg; Mol. Cell. Bio
l, 2,161, (1982)].

Hereinafter, the method for producing the DNA of the present invention and the recombinant DNA will be specifically described.

First, vector primer pSV7186 with mRNA and oligo dT
(Pharmacia) in an appropriate solution, for example, 50 mM Tris-HC1 buffer (pH 8.3), MgCl ₂ (for example, 8 mM), KCl (for example, 30 mM), dNTP (dATP, dTTP, dGTP, dCTP, for example, 2 m each)
M), dissolved in dithiothreitol (eg, 1 mM). A reverse transcriptase (eg, 1000 U / ml) [Takara Shuzo Co., Ltd.] is added thereto, and the mixture is added at a constant temperature (for example, 37 ° C.) and for a fixed time (for example,
Minutes). 3 ′ of the RNA-DNA duplex thus obtained
An oligo dC chain is added to the end.

First, the solution containing the RNA-DNA duplex is subjected to phenol / chloroform treatment, and, for example, an equal amount of 4MCH3C00Na is added, and ethanol precipitation is performed with twice the amount of ethanol.

The obtained pellet is mixed with a suitable solution, for example, 30 mM Tris-
HCl buffer (pH 6.8), CcCl (eg, 1 mM), dCTP (eg, 70 μM), cacodylic acid (pH 6.8) (eg, 140 mM
M), dissolved in dithiothreitol (eg, 0.1 mM).

To this solution, for example, terminal deoxynucleotidyl transferase is added at 1500 U / ml, and the time required for adding 8-12 oligo dC chains at a constant temperature (for example, 37 ° C.) (for example, 5 to 10 minutes) ) React.
Next, an RNA-DNA double-stranded vector primer to which an oligo dC chain has been added is added to a Tris-HCl buffer solution (pH 7.5) (for example, 10 m
M), MgCl ₂ (for example, 6 mM), NaCl (for example, 60 mM), dissolved in a solution, added with Hind III (for example, 20 U), and added at a constant temperature (for example, 37 ° C.) for a certain time (for example,
(0 min) (reaction) Mix the linker DNA (pSV1932) (Pharmacia) with the cDNA vector primer,
Tris-HCl buffer (pH 7.5) (for example, 20 mM), MgCl
₂ (for example, 4 mM), (NH ₄ ) ₂ SO ₄ (for example, 10 mM), KCl
(Eg, 0.1 mM), in a solution containing β-nicotinamide adenine dinucleotide (β-NAD) (eg, 0.1 mM) together with E. coli DAN ligase (eg, 6.7 U / ml) for a certain period of time (eg, 15 hours) ), For a certain time (for example, 12 ° C)
To react.

By this reaction, the cDNA and the linker DNA are circularized. In this reaction solution, dA was added to a final concentration of 40 mM.
TP, dTTP, dGTP, dCTP are added, and β-NAD (for example, final concentration of 0.
15 mM) E. coli ribonuclease H (for example, 17.7 U / ml)
To obtain a recombinant plasmid containing the complete double-stranded cDNA by replacing the RNA portion with DNA.

E. coli, for example, Escherichia coli DH1 strain, was transformed with the thus obtained recombinant plasmid, for example, by the method of Hanahan et al. [D.
J. Mol. Biol., 166, 557, (1983)]. Transformants can be selected by resistance to ampicillin, a marker.

The above-mentioned transformed Escherichia coli is immobilized on a nylon membrane, and is associated with a synthetic DNA probe having a DNA sequence predicted from the amino acid sequence of a known bluefin tuna growth hormone. (Proc.Natl.Acad.S
ci. USA, 72, 3961 (1975) Grunstein-Hogress method].
Probe DNA was obtained by the triester method (J. Am. Chem. Soc., 97, 732).
7 (1975)]. Selection using a synthetic DNA probe is performed by a method such as dot blotting, Southern et al. (J. Mol.
Biol., 98, 503 (1975)], and it is possible to identify a recombinant plasmid DNA having a gene complementary to bluefin tuna growth hormone mRNA by this method.

One example of a recombinant plasmid thus obtained is
pTTS339 (JP-A-1-104180). pTTS339 DNA
The sequence is shown in FIG. This plasmid can be used as a source of a gene encoding tuna growth hormone.

Production of Tuna Growth Hormone Polypeptide by Expression of Tuna Growth Hormone-Encoding DNA in Yeast: The DNA is cut out from a plasmid into which tuna growth hormone-encoding DNA has been incorporated, and this DNA is ligated to a vector DNA to obtain a set. The tuna growth hormone polypeptide can be produced by transforming the recombinant DNA into yeast, culturing the resulting transformed yeast to produce and accumulate the tuna growth hormone polypeptide in the medium, and collecting the tuna growth hormone polypeptide. it can. In addition to the above pTTS339 as a plasmid containing DNA encoding tuna growth hormone, pUES1
3, pUES13S-2 (both are described in JP-A-1-104180) is a preferred example.

Of pSH339 and pTTS339 used in the examples, pSH339 is p
Ec encoding tuna growth hormone N-terminal region of TTS339
It can be obtained by replacing the oR I-Cla I fragment with that of pUES13S.

Any vector DNA can be used as long as the inserted DNA can be expressed in yeast. To maintain the plasmid in yeast and to select for transformants in yeast and E. coli, the yeast replication origin 2 μM sequence and len2 (leucine non-required), Ap
A vector having a (ampicillin resistance) marker is used. Preferably, the promoter of the yeast repressible acid phosphatase gene (pPH05) [Richard. A. Kramer et al.
USA, 81, 367, (1984)] Phosphoglycerate kinase gene promoter (pPGK) [MJ
Dobson et al., Nucleic Acids Res, 10, 2625, (1982)],
Alcohol dehydrogenation yeast gene promoter (pADH1)
[Grant A. Bitter et al., Gene, 32, 263, (1984)], promoter of glyceraldehyde-3-phosphate dehydrogenase gene (pG3PDH) [Holland et al., J. Biol. Chem., 255, 2596,
(1980)], galactokinase gene promoter (pGAL1) [Johnston et al., Mol. Cell. Biol., 4, 1440, (198
4)] and a vector into which DNA can be inserted downstream
DAN is used.

A particularly suitable vector DAN is pAM82. pAM82 is the plasmid shown in FIGS. 2 and 3,
Its structure and manufacturing method are based on the method of Donggang et al. [Proc.
d. Sci. USA, 80, 1, (1983)].

Recombination between tuna growth hormone-encoding DNA and vector DNA can be carried out by using restriction enzymes used in general recombinant DNA techniques and their related enzymes.

As a specific example, in the case of pSL339, first, as shown in FIG. 2, Xh encoding tuna growth hormone from pSL339.
An ol-Pvu II digested fragment is obtained, and an Xhol-Pvu II fragment containing the yeast inhibitory acid phosphatase promoter is obtained from pAM82.

The above DNA fragment is ligated with T4 DNA ligase to obtain the recombinant plasmid pTGH82 shown in FIG. This plasmid has DNA encoding mature tuna growth hormone.

By performing the same treatment, a plasmid having a tuna growth hormone secretion signal can be prepared. For example, pS
Xhol-Pvu encoding tuna growth hormone from L339sp
Xhol-Pvu containing II digestion fragment and containing (pPH05) from pAM82
Obtain a II digested fragment. By binding the above DNA fragment with T4 DNA ligase, pTGH82sp having a tuna growth hormone secretion signal can be prepared (see FIG. 3).

The reaction conditions in the above recombination technique are generally as follows.

The digestion reaction of DNA with restriction enzymes is usually 0.1-50 μg.
The DNA was treated with a 1-150 mM (preferably 10-50 mM) Tris-HCl buffer (pH 6.0-9.0, preferably (pH 7.0-8.5), 0-200 mM).
M NaCl or KCl, 0-50 mM (preferably 5-20 mM)
The reaction is carried out at 10 ° C.-70 ° C. for 10 minutes-20 hours using 0.1-500 units of restriction enzyme (preferably 1-5 units per 1 μg) in a reaction solution containing MgCl ₂ . The reaction can be stopped usually by adding an equal amount of water-saturated phenol or inactivating the restriction enzyme by heating at 60 ° C. to 75 ° C. for 10 to 30 minutes.

The DNA binding reaction is carried out in a 1-100 mM (preferably 10-50 mM) Tris-HCl buffer (pH 6.0-9.0, preferably pH 7.0-8.
0), MgCl ₂ of 1-50 mM (preferably 5-20mM is), 0.1-10 mm
(Preferably 0.5-3 mM), 1-30 mM (preferably 5-10 mM) DT
The reaction is carried out in a reaction solution containing T using 0.1 to 1000 units of T4 DNA ligase at 1 to 30 ° C (preferably 4 to 16 ° C) for 20 minutes to 48 hours (preferably 4 to 24 hours).

The recombinant plasmid DNA generated by the ligation reaction is
If necessary, for example, a transformation method such as Hanahan [Hanahan: JM
ol. Biol., 166, 557, (1983)].

To isolate the DNA from E. coli containing the recombinant plasmid DNA, for example, a method such as Burnboim (Birnboin, etc.,
Nucleic Acids Res., 7, 1513, (1979)] or a method such as Holmes [Holmes et al., Analytical Biochemistyr, 11].
4, (1983)].

Confirmation of the cleavage site of the plasmid DNA by the restriction enzyme is performed by digestion with the restriction enzyme and then using agarose gel electrophoresis or polyacrylamide gel electrophoresis. Further, for determining the nucleotide sequence of DNA, a method such as Sanger using M13 phage and dideoxynucleotide (Sanger et al., Proc. Nat.
l.Acad.Sci.USA, 74,5463, (1977)] or Maxam
Gilbird et al. (Maxam Gilberd: Proc. Natl. Acad. S.
ci. USA, 74, 560 (1977)].

The tuna growth hormone polypeptide of the present invention can be produced as follows. Recombinant plasmid DNA (eg, pTGH8
2) using lto et al. [Lto et al., J. Bacteriol., 153, 16
3, (1983)], the yeast Saccharomyces cerevisiae SHY2 is transformed, and yeast containing pTGH82 is selected from leucine non-requiring colonies. This transformed yeast pTGH
82 / Saccharomyces cerevisiae SHY2 was sent to the Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology
7) has been deposited. By culturing this in a medium not containing inorganic phosphate, tuna growth hormone polypeptide can be produced in the culture.

Any medium can be used as long as it is suitable for growing yeast and producing tuna-grown formolin peptide.

Examples of carbon sources include glucose, glycerol, maltose, fructose, sucrose, lactose, mannitol, and sorbitol.
₄ Cl, (NH ₄ ) ₂ SO ₄ , yeast extract, corn stave liquor, polypeptone, meat extract, casamino acid, tryptophan, etc. Other nutrient sources include K ₂ HPO ₄ , KH ₂ PO
₄ , NaCl, KCl, MgCl ₂ , FeSO ₄ , vitamin B and the like can be used.

The cultivation is carried out by aeration and agitation culturing at pH 4.0-9.0, at a temperature of 15 ° C.-35 ° C.

Since the tuna growth hormone polypeptide accumulates in the cultured cells after 6 to 24 hours of culture, the cells are collected from the culture, and the polypeptide is collected from the cells according to a normal polypeptide extraction method.

Confirmation of tuna growth hormone polypeptide in the cells
Laemmli sample buffer [Lammli, Nature, 227, 680 (197
0)], and after lysis by heating, SDS-polyacrylamide electrophoresis [Lammli, Nature, 227, 680 (197)
0)] and by Coomassie brilliant blue staining.

Transformed yeast cells accumulating tuna growth hormone obtained by culturing can be used as such, or the cells can be crushed and added to a seafood feed to obtain a seafood feed having a growth promoting effect. .

Further, instead of the yeast cells or their crushed products, tuna growth hormone extracted from them may be added to the fish and shellfish feed. The amount of these yeast cells, their crushed products or tuna growth hormone is not particularly limited, and any amount may be used as long as the growth of fish and shellfish is promoted.

〔Example〕

 Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 Preparation of Recombinant Plasmid pTGH82 Encoding Mature Tuna Growth Hormone: 20 μg of plasmid pSH339 containing DNA encoding tuna growth hormone was ligated with 10 mM Tris-HCl buffer (pH 8.0), 7 mM.
M MgCl ₂ , ₂ mM _2- mercaptoethanol and 100 mM NaC
The resulting solution was dissolved in 50 μl of a solution containing l (hereinafter referred to as Y-100 buffer), added with 15 units of restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.), and digested at 37 ° C. for 2 hours. The reaction solution was extracted with phenol / chloroform and precipitated with ethanol, and the DNA fragment was treated with 30 μm of 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl 2.
₂ , dissolved in a solution containing 1 mM _2- mercaptoethanol and 33 μM dNTP (hereinafter referred to as Kleno-buffer), added with 5 units of Kleno-enzyme (manufactured by Takara Shuzo Co., Ltd.), and reacted at 37 ° C. for 30 minutes; The ends of this DNA were blunted. Then this T
DNA fragment and phosphorylated Xho I linker (Takara Shuzo Co., Ltd.)
Was added to 30 μm of 66 mM Tris-HCl buffer (pH 7.6), 66 mM Mg
It was dissolved in a solution containing Cl ₂ 10 mM DTT and 0.1 mM ATP (hereinafter referred to as a ligase buffer), and reacted with 100 units of T4 DNA ligase (Takara Shuzo Co., Ltd.) at 16 ° C. for 2 hours.

Using this reaction solution, Escherichia coli HB101 was transformed according to the method of Hanahan et al. To obtain an ampicillin-resistant strain, and the plasmid DNA was isolated by a method such as Burnboim to obtain pXH0339 shown in FIG. After digestion with EcoR I or Xho I restriction enzyme, agarose gel electrophoresis was performed to confirm the production of pXHO339.

Next, 10 μg of the thus prepared pXH0339 was
Was dissolved in a Y-100 buffer solution, and XHOI and NcoI (both from Takara Shuzo Co., Ltd.) were added in 20 units each, followed by digestion at 37 ° C. for 4 hours. From this reaction solution, 1 μg of a large plasmid fragment containing DNA encoding tuna growth hormone was obtained by low-melting point agarose gel electrophoresis.

On the other hand, the following DNA linker was synthesized (Takara Shuzo Co., Ltd.) to connect the vector DNA and the DNA encoding tuna growth hormone.

The phosphorylated synthetic linker and the plasmid pH obtained above
XO339 and XhoI-NcoI fragments were dissolved in 30 μg of ligase buffer, 100 units of T4 DNA ligase was added, and the reaction was carried out at 16 ° C. for 12 hours.

Using the reaction solution, Escherichia coli HB101 was transformed according to the method of Hanahan et al. To obtain an ampicillin-resistant strain, and the plasmid DNA was isolated by the method of Barnboim and the like to obtain pSL339 shown in FIG. After digestion with three types of restriction enzymes, EcoR I, Xho I and Nco I, the structure of pSL339 was confirmed by agarose electrophoresis.

Next, the plasmid pSL339,10
μg in 50 μl of Y-100 buffer, XhoI and pvuII
20 units of each (both from Takara Shuzo Co., Ltd.) were added, and digestion reaction was performed at 37 ° C. for 4 hours. From the reaction solution, 1 μg of a fragment (about 0.8 Kb) containing TDNA encoding tuna growth hormone was obtained by low-melting point agarose gel electrophoresis.

Separately, 10 μg of pAM82 was dissolved in 50 μ of Y-100 buffer,
Xho I and Pvu II (20 units each) were added, and digestion was performed at 37 ° C. for 4 hours. From this reaction solution, 5 μg of a fragment (about 10.6 Kb) containing a repressible acid phosphatase promoter (pPHO5), a marker for leu2, a 2 μM origin, and an ars gene was obtained by low-melting point agarose gel electrophoresis.

The Xho I-Pvu II fragment derived from pSL339 obtained above (about 0.8K
b) and an Xho I-Pvu II fragment (about 10.6 Kb) derived from pAM82 were dissolved in 30 μg of a ligase buffer, 100 units of T4 DNA ligase was added, and the reaction was carried out at 16 ° C. for 12 hours.

Using this reaction solution, Escherichia coli HB101 strain was transformed to obtain an ampicillin-resistant strain, and the plasmid DNA was obtained by the above method.
Was isolated to obtain pTGH82 shown in FIG. Nco I, Xho I,
After digestion with EcoR I and Pvu II, the structure of pTGH82 was confirmed by agarose gel electrophoresis.

Example 2 Recombinant plasmid encoding tuna growth hormone containing secretion signal: Dissolve 10 μg of pTTS339 in 50 μl of Y-100 buffer
A unit of EcoRI was added, and a digestion reaction was performed at 37 ° C. for 2 hours. After extracting the reaction solution with phenol / chloroform and precipitating with ethanol, in order to blunt the ends of the DNA, the DNA was dissolved in 30 μl of Kleno-buffer and 5 units of Kleno-enzyme was added.
The reaction was performed at 37 ° C. for 30 minutes.

This blunted DNA fragment and phosphorylated Xho I linker were
The resultant was dissolved in μ ligase buffer, 100 units of T4 DNA ligase was added, and the binding reaction was performed at 16 ° C. for 12 hours.

Using this reaction solution, Escherichia coli HB101 was transformed to obtain an ampicillin-resistant strain, the plasmid DNA was isolated, and the desired plasmid pXHO339sp was obtained.

10 μg of this pXHO339sp was dissolved in 50 μm of Y-100 buffer, and 10 units of XhoI and NcoI were added to each, followed by digestion at 37 ° C. for 4 hours. DNA encoding tuna growth hormone was obtained from this reaction mixture by low melting point agarose gel electrophoresis.
1 μg of a large plasmid fragment containing

The phosphorylated synthetic linker described in Example 1 and the plasmid pXHO339sp, XhoI-NcoI fragment obtained above were dissolved in 30 μl of ligase buffer, and 100 units of T4 DNA ligase was added.
The cyclization reaction was performed at 16 ° C for 12 hours.

Using this reaction solution, Escherichia coli HB101 was transformed to obtain an ampicillin-resistant strain, the plasmid DNA was isolated, and the desired plasmid pSL339sp was obtained.

10 μg of this pSL330sp was dissolved in 50 μm of Y-100 buffer, and 10 units of Xho I and Pvu II were added to each, followed by digestion at 37 ° C. for 4 hours. DNA encoding tuna growth hormone was obtained from this reaction mixture by low melting point agarose gel electrophoresis.
Was obtained (about 0.9 Kb).

This Xho I-Pvu II fragment derived from pSL339sp and the Xho I-Pvu II fragment derived from pAM82 obtained by the method described in Example 1 (about 1
0.6Kb) in 30μ ligase buffer and add 100 units
T4 DNA ligase was added, and a circularization reaction was performed at 16 ° C. for 12 hours.

Using this reaction solution, E. coli HB101 strain was transformed,
An ampicillin-resistant strain was obtained, the plasmid DNA was isolated, and the desired plasmid pTGH82sp was obtained. The structure of pTGH82sp is Nc
o After digestion with I, Xho I, EcoR I, and Pvu II, it was confirmed by agarose gel electrophoresis (see FIG. 3).

Example 3 Production of Tuna Growth Hormone Polypeptide by Yeast Containing pTGH82: Ito was performed using the recombinant plasmid pTGH82 obtained in Example 1.
Yeast Saccharomyces cerevisiae SH
The Y2 strain was transformed. The resulting leucine non-required transformant was transformed into a Burkholder minimal medium (0.2% (NH ₄ ) SO ₄ , 0.15% KH ₂ PO ₄ , 0.05% MgSO ₄ .7) containing 100 ml of inorganic phosphate.
H ₂₀ , 0.033% CaCl ₂・ 2H ₂₀ , 0.01ppm KI, trace elements, vitamins, 0.2% asparagine, 2% glucose, 25
μg / ml, histidine, 25 μg / ml tryptophan, 20 μg
/ ml uracil, pH 5.6) and at 30 ° C, OD610 = 1.
The cells were cultured until they reached zero. When OD610 = 1.0, culture medium is 5,000
The cells were collected by aseptically centrifuging at rpm for 5 minutes. The collected cells were placed in a Burkholder minimal medium (0.15% KH ₂ P
The suspension was suspended in 100 ml of a medium in which O ₄ was changed to 0.15% KCl) and cultured at 30 ° C. for another 24 hours. 24 hours later, 5,000 rpm, 5 minutes,
The cells were collected by centrifugation. The collected cells were suspended in 3 ml of 100 mM Tris-HCl buffer (pH 7.0) containing 0.1 mM phenylmethanesulfonyl fluoride (PMSF), and mixed with about 20 g of glass beads (B. Braun Melsungen AG). , 4
The cells were disrupted at 5 ° C. for 5 minutes, then suspended in a sample buffer of Laemmli, heated, and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, staining with Coomassie brilliant blue revealed a polypeptide band at a site with a molecular weight of 21,000. This electrophoresed gel is
Western blotting (Beisiegel et al., J. Biol. Ch.
em., 257, 13150, (1982)] and bound to an anti-tuna growth hormone antibody. From the results, it was found that pTGH82 / Sa-ccharomyces serevisiae SHY2 containing pTGH82 produced a tuna growth hormone polypeptide. Q Example 4 Production of tuna growth hormone polypeptide by yeast containing pTGH82sp: Example 2 Using the obtained recombinant plasmid pTGH82sp,
The yeast SHY2 strain was transformed according to the method described in o, etc. The obtained leucine non-auxotrophic transformant strain was cultured under the same conditions as in Example 3. After the culture, the cells were collected by centrifugation, and the cells were disrupted under the same conditions as in Example 3. Thereafter, the cells were suspended in a Laemmli sample buffer, heated, and then subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, staining with Coomassie Brilliant Blue revealed a polypeptide band at a site with a molecular weight of 21,000. This gel
When subjected to Western blotting, it bound to an anti-tuna growth hormone antibody. From this result, pTGH82sp
/ Saccharomyces cereviciae SHY2 pTGH82sp was found to produce tuna growth hormone polypeptide. pTGH82sp / SHY2 strain is pTGH82sp / Saccharomyces ce
revisiae SHY2 (Pierced Laboratories No. 2388) (FERM BP-23
88) has been deposited with the Research Institute of Microbial Industry and Technology.

Example 5 Measurement of Biological Activity of Tuna Growth Hormone Produced from Yeast: In the same manner as in Example 3, cells were collected from a 100 ml culture of pTGH82 / Saccharomyces cerevisiae SHY2 strain, and glass beads were used. The precipitate obtained by crushing by dialysis was dialyzed overnight at 4 ° C. against 50 mM Tris-HCl (pH 7.0) containing 8 M urea, and then subjected to an affinity column using tuna growth hormone as a ligand three times, to thereby obtain about 10 mg of tuna. I got growth hormone. This protein was confirmed to be single by SDS-polyacrylamide gel electrophoresis. The following examination was performed using the purified tuna growth hormone.

Thirty juvenile bluefin tuna having a body length of about 15 cm and a body weight of about 100 g were divided into two groups and placed in two sets of water tanks at a water temperature of 25 ° C. Seawater was circulated and water stains were removed by filter filtration. Food was given twice daily at 10 am and 5 pm until satiety. The tuna growth hormone obtained above was dissolved in 0.5 μg, 100 μ of physiological saline and injected intraperitoneally four times every 5 days in one set. The remaining one set was intraperitoneally injected with 100 μ of physiological saline four times every 5 days to serve as a control. Every 5 days, body length and weight gain were measured. The result is shown in FIG.

Example 6 Efficacy of Yeast Cells Producing and Accumulating Tuna Growth Hormone in Cells as Feed for Fish and Shellfish: In the same manner as in Example 3, cells were cultured from a culture of pTGH82 Saccharomyces cerevisiae SHY2 strain 20. Was collected, crushed using Dinosyl (manufactured by Willy AB), and then lyophilized to obtain dried yeast cells that produced and accumulated tuna growth hormone. The following examination was performed using the dried yeast cells.

Twenty rainbow trout juveniles having a body length of 7 to 8 cm and a weight of about 10 g were divided into two groups, placed in two sets of water tanks at a water temperature of 20 ° C., and reared for 28 days. Feed twice daily, 9am and 4pm, initial feed P2
0.5+ 0.5 g of dried yeast cells / tail were fed instead of the normal diet. The remaining one set was bred on a normal diet and used as a control. Body length and weight gain were measured every 7 days. The results are shown in FIG.

In the case of the set fed with the dried yeast cells that produced and accumulated tuna growth hormone in the cells, the body length and weight were significantly increased compared to the control set. The effectiveness as was confirmed.

From the above results, it was confirmed that the tuna growth hormone polypeptide produced by yeast had a growth promoting effect.

Reference Example 1 Preparation of recombinant plasmid pSH339 containing DNA encoding mature tuna growth hormone: 10 μg of pTTS339 encoding tuna growth hormone was
100 mM Tris-HCl buffer (pH 7.5), 7 mM MgCl ₂ , 50 mM N
aCl, a solution containing 7 mM 2-mercaptoethanol (hereinafter referred to as Y
-100 buffer), 10 units of each of EcoRI and ClaI were added, and a digestion reaction was carried out at 37 ° C for 4 hours. From this reaction solution, a fragment containing the DNA encoding tuna growth hormone C-terminal side (approximately
3.3 Kb) 1 μg was obtained.

Separately, 10 μg of pUES13S was dissolved in 50 μm of Y-50 buffer, each unit of EcoRI and ClaI was added, and a digestion reaction was performed at 37 ° C. for 4 hours. From this reaction solution, 1 μg of a fragment (about 0.2 Kb) containing DNA encoding the N-terminal side of tuna growth hormone was obtained by low-melting point agarose gel electrophoresis.

This pTTS339-derived EcoR I-Cla I fragment (about 3.3 Kb) and p
The UES13S-derived EcoR I-Cla I fragment (about 0.2 Kb) was dissolved in 30 μg of ligase buffer, 100 units of T4 DNA ligase was added, and a circularization reaction was performed at 16 ° C. for 12 hours.

Using this reaction solution, E. coli HB101 strain was transformed,
An ampicillin-resistant strain was selected, the plasmid DNA was isolated, and the desired plasmid pSH339 was obtained. The structure of pSH339 is
After digestion with NcoI, EcoRI, and ClaI, it was confirmed by agarose gel electrophoresis (see FIG. 5).

〔The invention's effect〕

According to the present invention, fish growth hormone polypeptide containing no endotoxin can be produced in large quantities by culturing yeast having recombinant DNA. Since the produced growth hormone was found to exhibit a fish growth promoting effect, it is expected to be applied to the aquaculture industry.

[Brief description of the drawings]

FIG. 1 shows the entire nucleotide sequence of the tuna growth hormone gene in plasmid pTTS339, FIG. 2 shows the construction of plasmid pTGH82, FIG. 3 shows the construction of plasmid pTGH82sp, and FIG. FIG. 5 shows the increase in body length and body weight when growth hormone was administered, and FIG. 5 shows the body length and body weight when the dried tuna growth hormone-producing yeast cells obtained according to the present invention were mixed into a bait and administered to red sea bream larvae. FIG. 6 shows the increase, and FIG. 6 is a construction diagram of plasmid pSH339.

──────────────────────────────────────────────────の Continuing on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 865) (72) Inventor Michio Nonaka 3-2-9 Tsukishima, Chuo-ku, Tokyo Ocean Fisheries Inside Ocean Research Institute Co., Ltd. (72) Inventor Yoshiharu Inoue Rabbit Road 2A-6 Uta-shi, Uji-shi, Kyoto (72) Kosaku Murata Inventor 3-35, Hitsukawa-cho, Yamashina-ku, Kyoto-shi, Kyoto (72) Inventor Hikaru Kimura Wakamiya-dori, Shimogyo-ku, Kyoto-shi, Kyoto (56) References JP-A-1-104180 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A yeast having a recombinant DNA into which a DNA encoding tuna growth hormone containing the amino acid sequence shown in FIG. 1 has been cultivated in a medium.
A method for producing fish growth hormone, comprising accumulating a tuna growth hormone polypeptide and collecting the tuna growth hormone polypeptide from the culture. 2. The method for producing fish growth hormone according to claim 1, wherein the yeast is a yeast belonging to the genus Saccharomyces. 3. The method for producing fish growth hormone according to claim 2, wherein the yeast belonging to the genus Saccharomyces is Saccharomyces cerevisiae. 4. The amino acid sequence shown in FIG.
A feed for fish and shellfish comprising a yeast cell which has accumulated a tuna growth hormone polypeptide containing amino acids 1 to 187 or a crushed product thereof.