JPS61210100A - Novel growth hormone polypeptide for fish - Google Patents

Abstract

NEW MATERIAL:The growth hormone polypeptide for fish, having the peptide sequence of formula. USE:Fish-growth promoting agent for pisciculture. PREPARATION:For example, the pituitary gland of Oncorhynchus keta is disinte grated and solubilized in a solution containing guanidium thiocyanate, and added to an EDTA solution. The mixture is centrifuged to recover the LRNA as a precipitate, which is dissolved in a concentrated solution of NaCl, etc., and passed through a column of oligo(dT)cellulose to obtain an mRNA having poly(A). A cyclic cDNA is synthesized from the mRNA, a vector primer and a linker DNA, and Escherichia coli is transformed with the cDNA to obtain a mutant containing a recombinant DNA integrated with a DNA coding the fish growth hormone. The mutant is cultured in a nutrient medium to obtain the objective polypeptide having the structure of formula.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention uses a DNA encoding a novel fish growth hormone polypeptide, a recombinant DNA incorporating the DNA, a microorganism containing the recombinant DNA, and the microorganism. This invention relates to a method for producing a fish growth hormone polypeptide. Fish growth hormones are expected to have wide applications in the fish aquaculture industry.

BACKGROUND OF THE INVENTION Mammalian growth hormones are produced in the pituitary gland, and their activity and structure are known. for example,
Human growth hormone was published in the Journal of the American Chemical Society (J. Am.) by U. J. Lewis et al.
Chem, Soc, ), 80, 4429 (195
g), A, 1st + Bartley (A, S, Har
Biochemical Journal (Bi
ochem, J.).

100.754 (1966), /- Goichi Lee
(Archives on 2 by (: Hl, l) et al.
Iokeminutritando High-Ovenox (Sublumenoto) (, Ar[, h, Biochem, B+op
hys, (Suppl,), i, soil, 327 (+q6
2>.

Regarding fish growth hormones, there have been many reports that have been shown to be simple and elegant, but there are only a few that are reliable in terms of their physiological activity and protein chemical properties. Examples of reliable reporting include:

Isolation example from tilapia: F.W.F.
S, ltl, Farmer) et al.
Comparative Endocrinology (Gen.

Comp, 1 Endocrin, 30.91 (
1976) Isolation example from sturgeon “Niss Dove IJ,
- (S, W, Farmer) et al., Endocrinology,
108.377 (1981) Isolation example from carp: A.F. Tuck (A, F.).

Cook et al., General and Comparative Endocrinology (Gen, Comp, Endo)
crin,).

50, 335 (1983).

On the other hand, regarding the growth hormone gene of clear mammals, the rat growth hormone gene [B.H.
H, Seeburg) et al.: Neich...? -(Nat,
ure) 270486 (+977)), tsu/and pig growth hormone JJNA), 2. 37 (1
983)], human growth hormone gene [G.A.
Mart+al, LA et al., ScienceC, 602 (1979)
] are already known, and the fish growth hormone gene has also been isolated by the present inventors [
Patent application No. 59-134536 and No. 59-213360
:]. The fish growth hormone polypeptide encoded by this gene is a salmon growth hormone polypeptide previously prepared from the salmon pituitary gland by Kawauchi et al., and which has also been confirmed to have a growth-promoting effect in teleosts [Patent Application No. 1983] -68670:], N-terminus (40), C-terminus (
The amino acid sequences of 21 amino acids were completely identical.

Problems to be Solved by the Invention Fish growth hormone has a growth-promoting effect on fish, and is therefore useful as a composition of fish feed. However, the supply of the hormone collected from the pituitary gland of fish is limited. . Therefore, it is desired to develop a method for supplying fish growth hormone in large quantities at low cost.

Means for Solving the Problems The present inventors conducted research on a method for producing fish growth hormone by recombinant DNA technology. the result,
We succeeded in collecting NA from a single batch of fish growth hormone polypeptide, which can be used for the production of fish growth hormone, and in producing recombinant DNA and microorganisms containing it. That is, from the salmon pituitary gland to the metsenger RN.
A (mRNA) is extracted, and complementary RNA (mRNA) is extracted.
cDNA), then synthesized a DNA probe corresponding to the amino acid sequence near the N-terminus of salmon growth hormone, and selected a cDNA that hybridized with this DNA to clone the salmon growth hormone gene. It was successful and I have already applied for a patent (Patent application 1982-1)
34536 and 59-213360). Furthermore, by examining several salmon growth hormone genes in detail, we discovered that there was another gene that had a high correlation with the one for which we had previously applied for a patent, and determined the entire base sequence of its cDNA. The inventors further conducted research and created a recombinant DNA that incorporates the DNA encoding the new salmon growth hormone.
The present inventors have discovered that salmon growth hormone polypeptide can be produced and accumulated in significant amounts in the culture by culturing microorganisms containing the same, and have completed the present invention.

The present invention will be explained in detail below.

The present invention provides novel fish growth hormone polypeptides, particularly polypeptides having the peptide sequences shown in Table 1. The polypeptide can be produced using silk recombinant DNA technology as described below.

That is, +nRNA of fish growth hormone is used as a template to prepare a DNA (cDNA>) that exhibits eye-catching complementarity to the mRNAΔ, and a recombinant plasmid incorporating the cDNAΔ is prepared.Furthermore, the recombinant plasmid into a host microorganism. The DNA and the recombinant plasmid can be used for the amplification of novel growth hormone genes, especially in bacteria such as E. coli. The microorganism carrying the recombinant plasmid can be used to It is useful for producing a novel growth hormone polypeptide in large quantities at low cost.

Therefore, the present invention provides a DNA encoding a novel fish growth hormone polypeptide, a recombinant DNA incorporating the DNA, a microorganism containing the recombinant DNA, and a method for producing a novel growth hormone polypeptide using the microorganism. provide.

The DNA and recombinant plasmid of the present invention are prepared by the following general method.

Total RNA was prepared from the pituitary gland of black salmon, and this was added to oligo(dT) cellulose.
ose:] By passing it through a column, polyadenylic acid [Bo! J(A) :RNAC BoIJ(A)RN with 1
A) is separated.

Using this po'J (A) RNA as a template, double-stranded DNA is synthesized using reverse transcriptase. Silk recombinants are produced by inserting the synthetic DNA into vector DNA such as E. coli plasmid DNA using in vitro DNA recombination techniques.

Next, the method for producing the invented DNA and recombinant plasmid will be specifically explained.

The pituitary gland is removed from a captured black-spotted salmon and immediately frozen in liquid nitrogen. This frozen pituitary gland was treated with guanidium inthiocyanate (guan1d+um+5oth).
iocyanate), crush, and solubilize. Then C5Cj! The solution layer is layered, and after ultracentrifugation, total cytoplasmic RNAΔ is obtained as a precipitate. Alternatively, only RNA can be precipitated and recovered by adding Li(,j') to the guanidium inthiocyanate solubilized product.

The extracted RNA was treated with high salt concentration of NaCl or KCj7 (
For example, oligo(dT) dissolved in a 0.5 M) solution
m having a pore IJ (A) by passing through a column of cellulose
Adsorb RNA onto the column. Water, 10m M) I
The poly(Δ) bearing mRNA is isolated by elution using a low salt solution such as JSu HCl buffer.

Below, Okayama-Berg (Okayama-Berg)
) Method [Okayama 7-77 de Barb (Okayama
& Berg), Mol.
161 (1982)], synthesis into cDNA and its integration into a vector are performed.

First, synthesize vector brimer. As a vector+
For example, pCDV 1 may be dissolved in a suitable solution such as Tris-H (l buffer (e.g. pf 17.5.10mM),
treated with Kpn I in a solution containing M g C12 < e.g. 6mM), NaCR (e.g. 10mM);
Cut pCDV 1 at the Kpn 1 site.

This DNA was added to a Tris-HCl buffer (e.g. pH 6,
8,30mM), sodium cacodylate (for example] 40mM), CoCj! 2 (e.g. 1mM), dithiothreitol (e.g. 0.1mM) and dT
TP (e.g. 0.25mM) with terminal deoxynucleotidyl transferase at a constant temperature
The vector DNA is incubated at 37° C. for a certain period of time (for example, 20 minutes) to add approximately 60 thymidyl residues to both 3' ends of the vector DNA. Further, this DNA was diluted with Tris-H (l buffer (e.g. pH 7.5, 10mM
), MgCj! 2 (e.g. 6mM), NaC
j! EcoRI in a solution containing (e.g. 100mM)
After cutting, low melting point agarose gel electrophoresis [Lars W+eslander]
: Analytical Biochemistry (Anal
ytical Biochemistry, ) 93
.

305 (1979)), and a fragment of about 3.1 kilobases is recovered. The DNA is then dissolved in a high salt concentration (e.g., 0.5 M) solution of NaCR or KCl and passed through a poly(dA) cellulose column to obtain a poly(dA) cellulose column.
Only vector primer molecules having (T) are adsorbed onto the column. Elute with a low salt solution such as water, 10mM) Lis-HCl buffer to isolate only the poly(T)-attached vector primer molecules.

Next, linker DNA is synthesized. For example 1-ILLD
The NA is dissolved in a suitable solution, such as Tris-HCl buffer (e.g. I)H7,5,I OmM), MgC12 (
For example, 6mM), NaCf! (e.g. 50mM)
pL] is treated with PstI in a solution containing pL to cleave the PstI site. This DNA was replaced with dG instead of dTTP.
The procedure is the same as for vector primer synthesis except that TP is added, and about 15 oligo (dG) chains are added. The DNA is dissolved in a suitable solution such as Tris-H (l buffer (e.g. pH 7, 5, 10mM), Mg
C12 (e.g. 5mM).

In a solution containing NaCl (e.g. 60mM) @i n
Cut with dIIr. DNA fragments of approximately 0.5 kilobases are fractionated by agarose gel electrophoresis and recovered using DEAE paper. In this way, linker DNA is obtained.

The PO obtained as above! cDNA synthesis is performed using I(A) RNA, vector primer, and linker DNA. Poly (A) RNA and vector primer DNA were added to Tris-HCl buffer (e.g. pH 8, 3, 50mM).
, MgCL (e.g. 8mM), KCI! (e.g. 301TIM), dithiothreitol (e.g. 0
．． 3mM), dATP.

dTTP, dCTP, dGTP (e.g. 2mM each
) in a solution containing reverse transcriptase at a constant temperature (e.g. 37
℃) for a certain period of time (for example, 40 minutes). At the 3' end of the RNA-DNA duplex thus obtained, dTTP was added.
is changed to dCTP (dT
) Approximately 15 oligo(dC) chains are added under the same conditions as those for adding the chains.

This DNA was added to a Tris-HCl buffer (e.g. pH 7,
5,10mM), MgCL<eg 5mM).

in a solution containing NaCR (e.g. 60 mM) 1 i
Cut with ndIll. The previously prepared linker DNA was mixed with this DNA, and a Tris-HCl buffer (e.g. pH 7, 5, 20mM>), MgCL (e.g. 4mM
), (NH-)250- (e.g. 10mM), KC
l2 (e.g. 0.1 lVl'), β-nicotinamide, gamma denin dinucleotide (β-NAD) (e.g. 0
．． lrr+M) with E. coli DNA ligase for a certain period of time (for example, 16 hours) at a constant temperature (for example, 12° C.). Thus cDNAΔ
and linker DNA are circularized. Add dATP and dTTP to this reaction solution.

Add dGTP and dcTP to a final concentration of 40 μM, add E. coli DNA IJ gauze, E. coli DNA polymerase ■, and E. coli ribonuclease H, and add RN.
By converting the A part to DNA, a complete duplex c
A recombinant plasmid containing DNΔ is obtained.

Using the thus obtained recombinant plasmid, Escherichia coli, for example, Escherichia coli C600SF8 strain, is grown using Scots.
tt) et al.'s method [Katsuya Shigesada: Cell Engineering 2.616 (1
983) ).

Since the ampicillin resistance gene is present on the recombinant plasmid obtained in Section L, the transformed E. coli exhibits ampicillin resistance. The following method uses these ampicillin-resistant (A p') strains to generate a new recombinant plasmid DNA containing a gene that is complementary to fish growth hormone mRNA.
It is commonly used to select strains harboring . That is, the transformed strain obtained above was immobilized on a nitrocellulose filter, and allowed to associate with a synthetic NA probe having a DNA sequence predicted from the known amino acid sequence of chum salmon growth hormone. Select [Grunste+n
-) 1ogness) Method, Proceedings on the National Academy of Sciences on Scienne,
(Proc, Natl, Acad, 5ci),,
USA, 72°3961 (+975)]. Probe DNA was obtained using the usual triester method [Journal on American Chemical Society (J, Am,
Chem, Sac, ), 97.

7327 (1975> ).Synthesized NA
Southern selection by probe
[Journal on Molecular Biology (J, 1Jol, Biol, ), 98.50]
3 (1975)), and by this method, recombinant plasmid DNA having a gene complementary to chum salmon growth hormone mRNA can be identified.

An example of a recombinant plasmid obtained in this way is p
scH14. This plasmid can be used as a source of DNA encoding salmon growth hormone.

Production of fish growth hormone polypeptide by expression of DNA encoding salmon growth hormone in microorganisms: Cutting out the DNA from a plasmid containing the DNA encoding salmon growth hormone, integrating it into vector DNA, and the resulting recombination Salmon growth hormone polypeptide can be produced by introducing the body DNA into a microorganism and culturing the resulting transformant to produce and accumulate salmon growth hormone polypeptide in the culture. Salmon growth hormone polypeptide can be produced by collecting this.

A suitable example of a plasmid containing DNA encoding salmon growth hormone is the above pSGH14.

Any vector DNA can be used as long as the inserted DNA can be expressed in microorganisms↓)
Can be used. Preferably, a suitable promoter, such as the tryptophan (trp) system, lactose (
It has a promoter such as Iac) system or PL system, and DNA can be inserted downstream of it, and an appropriate distance between the internal Nyain Dalgarno sequence (hereinafter abbreviated as SD sequence) and the translation start codon (ATC), for example. Vector DNA adjusted to 6 to 18 base pairs is used. A specific example of suitable vector DNA is plasmid ρGELI. pGELl is a plasmid shown in Figure 3, and the Escherichia coli containing it is Escherichia coli.
It has been deposited as IGELI (FERM BP-629) at the Institute of Microbial Technology (Feikokuken), Agency of Industrial Science and Technology on October 6, 1981. Recombination between DNA encoding a polypeptide and vector DNA can be performed using a general recombinant DNA technique in which both DNAs are digested using restriction enzymes and then ligated using T4DNA ligase.

In the case of pSGH14 and pGε21 shown as specific examples, two stages of construction are performed as shown in FIG. That is, p
an Mbo II-Pvu II fragment encoding the vicinity of the N-terminus of salmon growth hormone mature peptide from SGH14;
Pvu containing the remainder to the cDN and a portion of the vector
- Obtain the old nd[II fragment separately. On the other hand, the following synthetic NA clinker is produced.

The above DNA fragment and synthetic NA clinker are combined into T4DNA!
The recombinant plasmid ρSfJ II 89 shown in FIG. 3 is obtained by ligating with J gauze.

Next, an old nd[-BamHI digested fragment encoding salmon growth hormone mature peptide is obtained from ρ5GIIIB9, and a HindI[[-BamHI digested fragment containing a tryptophan promoter is obtained from pGEL1. The above two DNA fragments are joined using T4 DNA IJ gauze to obtain the recombinant plasmid pSGH]Ic2 shown in FIG. This plasmid has a region encoding mature salmon growth hormone linked downstream of the tryptophan promoter.

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

The digestion reaction of DNA with restriction enzymes is usually 0.1 to 20
μg of DNA to 2-200mM (preferably 10-40mM)
mM) of trix-HCj! (pH 6.0-9.5 preferably pH 7.0-8.0), 0-200mM NaC
j! , 2-30mM (preferably 5-10mM) Mg
'CI1. restriction enzyme in a reaction solution containing 0.1 to 100
units (preferably 1 to 3 units per 1μg of DNA)
for 15 minutes to 24 hours at 20 to 70°C (the optimum temperature varies depending on the restriction enzyme used). The reaction is usually stopped by heating at 55 to 75° C. for 5 to 30 minutes, but a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

Purification of the DNA fragments generated by restriction enzyme digestion was performed using low melting point agarose gel electrophoresis [L.
L, Wieslander): Analytica/lz・
Biochemistry (Analytical Bioc)
Hemistry) 98.305 (1979), hereinafter referred to as the LGT method], polyacrylamide gel electrophoresis, etc.

The DNA fragment binding reaction is carried out using 2-200mM (preferably 10-40mM) Tris-HCl (pH 6,1-40mM).
9.5, preferably pH 7.0-8.0), 2-20m
M (preferably 5-10mM) of MgCL, 0.1-
In a reaction solution containing 10mM (preferably 0.5-2,0mM) of ΔTP, 1-50mM (preferably 5-10mM) of dithiothreitol, 0.3-10 units of T4DNA ligase was used, and 1 ~37°C (preferably 3-20°C) for 15 minutes to 72 hours (preferably 2-2
0 hours).

The recombinant plasmid DNA produced by the ligation reaction is
If necessary, the transformation method of Cohen et al.
Cohen (S, N.) et al.: Proceedings on the National Academy of Sciences (Proc. Natl. Acad. Sci.)
.

2110 (1972)) into E. coli.

Isolation of recombinant plasmid DNA from Escherichia coli containing recombinant plasmid DNA can be carried out by the method shown in Example 1 described later or by the Birnboil method D (H.
) et al.: Nuclei & Research (Nuclei) et al.
c^cids Res, ) Yu, 1513 (1979
)) etc.

After digesting plasmid DNA with 1 to 10 types of restriction enzymes, the cleavage site is examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

Furthermore, when it is necessary to determine the base sequence of DNA, the Maxam-Gilbert method [Procedures on the National Academy of Sciences: x, (Pr.
oc, Natl, Acad, Sci, )
, 74. 560 (1977)) or the Sanger method using M13 phage [Sanger et al.
National Academy on Science (Proc.
, Natl, Acad, Sci, ) USA
.

74, 5463 (1977); Ama-Jam (^me
M13 Cloning and Sea Fencing Handbook (cloning and sea fencing)
sequencing handbook)).

Recombinant plasmid DNA can be produced under the conditions described above.

The fish growth hormone peptide of the present invention can be produced as follows.

That is, a plasmid (e.g. ρ5GII n ['2
) to transform E. coli with -12HBIOI,
pSGHn C2 from ampicillin-resistant colonies
Select E. coli that has the following. By culturing E. coli containing pSGHnc2 in a medium, fish growth hormone polypeptide can be produced in the culture.

As the medium used here, either a synthetic medium or a natural medium can be used as long as it is suitable for the growth of E. coli and the production of fish growth hormone polypeptide.

Carbon sources include glucose and fructose.

Lactose, chrycerol, mannitol, sorbitol, etc. are nitrogen sources such as NH, C12゜(NH,)
2SO, casamino acids, yeast extract, polypeptone, meat extract, batatotryptone, corn staple liquor, etc.Other nutritional sources include K2HP 04,
KH2P 04 , NaCl.

Mg5O, bi9min B+, MgCβ2, etc. can be used.

Culture at pH 5.5-8.5. Culture is carried out at a temperature of 18 to 40°C by aeration and stirring.

Chum salmon growth hormone polypeptide accumulates in the cultured cells after 5 to 90 hours of culture, so the cells are collected from the culture, treated with lysozyme, and repeatedly frozen and thawed to crush the cells. After centrifugation, polypeptides are collected from the resulting supernatant according to a conventional polypeptide extraction method.

In addition, the polypeptide can be detected by directly using the culture medium and bacterial cells.
(Laemmli) Sample Ha Sofa [Laemmli (Laemmli), Nature (Nature)
, 227, 680 (1970)), 5DS-Polyacryl 1 Midgel [Rem'J (La
The method of Emmli) is applied to the above-mentioned document] and carried out by Cumanbu IJ 11'7' blue staining.

Examples of the present invention are shown below.

Example 1. Bo IJ (A) RN from the pituitary gland of salmon
Preparation of A.

Guanidium thiocyanate-cesium chloride method [Maniatis
) et al., eds., Molecular Cloning (Molecular Cloning)
lar Craning), p195. :] - Co1d Spring, Harbor
Published by Harbor).

Katsuya Shigesada, Cell Engineering, 2, 616 (19g3))
RNA having poly(Δ) was prepared as follows.

2g of frozen pituitary glands (approximately 30 individuals) were mixed with 4M guanidium thiocyanate, 0.5% sarcosine, 5m
M sodium citrate (p)-IT> and 0. I
The mixture was solubilized by crushing it with Teflon homogenizer (5 rpm) in a solution IQml consisting of Mβ-mercaptoethanol. This homogenate was passed through an 18G syringe needle several times to disrupt the DNA. 5.7MC5CA, 0, LM E
1.2 ml of each DTA (pH 8) solution was dispensed into ultracentrifuge tubes, and the homogenate was layered thereon. ) lita
chi RP S 40 units - 35,000 in Ku
After centrifugation at rpm for 1.5 hours, RNA was collected as a precipitate. The RNA precipitate was dissolved in IQml of Tris-H (1 (pH 8,0) solution containing 1mM EDTA, extracted with phenol-chloroform, and then recovered by ethanol precipitation. Approximately 11 g of the obtained RNA was dissolved in 10mM Tris-H (pH 8,0). (1 (pH 8,0) and 1mM EDTA
It was dissolved in 1 ml of a solution consisting of

Incubate at 65°C for 5 minutes, Q, 1 ml of 5MN
aCl1 was added. The mixture was subjected to oligo(dT) cellulose column (P-L Biochemicals) chromatography (column volume > 0.5 ml. mRNA with adsorbed poly(Δ) was 10 mM).
Lis-HC1(p)17.5> and 1mM EDT
It was eluted with a solution consisting of A and Q, and fractionated into 2 ml portions. 3~
Both fifth portions were collected to obtain about 10 μg of mRNA containing poly(Δ).

Example 2. cDNA synthesis and insertion of the DNA into a vector: t Kayama-Ber;
) method [Molecular and Cellular Biology (Mol, Ce1l, Biol, ), 2
cDNA Δ was synthesized and a recombinant plasmid incorporating it was constructed according to the following method.

An outline of the process is shown in FIG.

pcDVl (Okayama & Hague)
ma & berg): Molecular &amp;
Cellular Hiology (Mol, Ce1l, Bio
l, ), 3.280 (1,983) : l 4
00 μg in 10 mM Tris-HC (1(T) 87.
5), 6 m M M g CR2 and lQm
In addition to 300 μl of a solution consisting of MN a Cl, 500 units of Kpnl (from Takara Shuzo Co., Ltd.; all restriction enzymes not mentioned below are manufactured by Takara Shuzo Co., Ltd.) were added, and the mixture was incubated at 37°C for 60 minutes.
After a time reaction, the plasmid was cleaved at the Kpn1 site. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation.

Approximately 200 μg of the Kpn i-cleaved DNA was added to 40 m
Sodium dilate, 3QmM) Lis-HCJ' (
pH 6,8), 1 mM CaCf! , and 0
．． 1mM dithiothreitol (hereinafter abbreviated as DTT)
dT in a buffer solution (hereinafter abbreviated as TdT buffer) consisting of
Add 200μβ of a solution containing TP to 0.25mM, and add 81 units of terminal deokine nucleotidyl transferase (hereinafter abbreviated as TdT).
(PLB+Ochemicals vJ) was added and reacted at 37°C for 11 minutes. Here, K of pCDVI
Approximately 67 poly(dT) chains are located at the 3' end of the pn I cleavage site.
Added. Approximately 100 μg of poly(dT) chain-added pcDVI DNA was recovered from the solution by phenol-chloroform extraction and ethanol precipitation. The DNA was added to 10mM) Lis-HCJ! (pl+7.5), 6mM
In addition to 150 µp of a buffer consisting of Mg (1!, 100 mM NaCl), 360 units of EcoRI were added and reacted for 2 hours at 37°C. After processing the reaction product by the LGT method, a DNA of approximately 3.I K b was added. Collect the fragment and add approximately 60 μg of BoIJ (dT) 1iJ1-added pCDV.
I got 1. The DNA was diluted with 10mM) Lis-HC1p)18
．． Solution 5004 consisting of 0> and 1mM EDTA
After incubating at 65°C for 5 minutes, cool on ice and incubate for 5 minutes.
04 of 5M NaCl2 was added. The mixture was diluted with oligo(dA
) Cellulose column manufactured by Collaborative Research Co., Ltd.) was subjected to chromatography. Poly(dT) with sufficient chain length is adsorbed onto the column and added to 10mM) Lis-HCR (
It was eluted with a solution consisting of pH 8.0) and 1 mM EDTA, and pcDVl (
27 μg of the vector primer (hereinafter abbreviated as vector primer) was obtained.

Next, linker DNA is prepared.

pLl (Okayama & Barb)
Berg): Molecular and Cellular
Bioloshi (Mol, Ce1l, Biol,),
3.280 (1983): ] Approximately 14 μg to 10 mM
Tris-HCj! (pH 7,5).

5mM MgCC and 50mM NaCj! In addition to 200 μl of buffer consisting of
P in pLIDNΔ was added and reacted for 4 hours at 37°C.
It was cleaved at the st1 site. After the reaction product was extracted with phenol-chloroform, ethanol precipitation was performed, and about 13 μg of pLIDNA cut with Pstl was recovered. Approximately 13 μg of the DNA was added to TdT buffer with dGT at a final concentration of 0.25 mM.
In addition to 50 μl of a solution containing P, T d T (
54 units of P-L Biochemicals) were added and incubated at 37°C for 13 minutes to transform the Pst of pLl.
Approximately 14 (dG) chains were added to the 3' end of the l cleavage site.

D by ethanol precipitation after phenol-chloroform extraction
NA was collected.

100 μj of the DNA! lOmM) sy, -HCj!
(pH7,5>, 6 m M M g Cj!
In addition to 100 μl of a buffer consisting of 2 and 60 mM NacJ, 80 units of old nd■ were added and incubated at 37° C. for 3 hours, and 1) LIDNA was cleaved at the Hind111 site. The reaction product was fractionated by agarose gel electrophoresis, and a DNA fragment of about 0.5 Kb was separated using the DEΔE paper method [Dretze port], Analytical Biochemistry (Anal, Bioche).
m, ).

112, 295 (1981)) and oligo (
dG) Chained linker-DNA (hereinafter simply referred to as linker-D)
(abbreviated as NA) was obtained.

Approximately 2 μg of BoIJ(A) RNA and approximately 1.4 μg of vector primer prepared above were added to 50 mM Tris-HCl (p
H8,3), 8mM MgCA2.30mM KC
ji! , 0.3mM DTT, 2mM dNTP (d
ATP, dTTP, dGTP and dCTP) and 1
0 units glue bonuclease inhibitor (P-L
Biochemicals) solution 22.3
The mixture was dissolved in μl, 10 units of reverse transcriptase (Seikagaku Corporation) was added, and incubated at 37° C. for 40 minutes to synthesize NA complementary to mRNA8. The reaction product was extracted with phenol-chloroform and precipitated with ethanol.
- Vector primer DNA with added DNA double strand was recovered. The DNA was treated with 66μM dCTP and 0.2μ
Dissolved in 20μβ of TdT buffer containing gpoIJ(A),
14 units of TdT (PL Biochemica
ls) and incubate for 8 minutes at 37°C.
Twelve (dC) strands were added to the 3' end of the DNA.

The reaction product was extracted with phenol-chloroform, and the cDNA-vector primer DNA with the (dC) strand added was recovered by ethanol precipitation. The DNA was dissolved in 1QmM) Lis-HCl (pH 7,5), 6mM g CI 2
and 60mM NaCj! A liquid consisting of 400 μj!
Add 20 units of ) l i ndnI to 37
The ink was incubated at °C for 2 hours and cut at the Hind ■ site. The reaction product was extracted with phenol-chloroform and precipitated with ethanol to obtain 5 pmole of (dC) chain-added cD.
NA-vector primer DNA was obtained. The DNA0.
081) mole and the linker DNA 0.16 above
pmole 10mM) Lis-HC1 (pH 7,5)
,'0. IM NaCj! and 40 μl of a solution consisting of 1 mM EDTA at 65°C.

The cells were incubated at 42°C and 0°C for 10 minutes, 25 minutes, and 30 minutes, respectively. 20mM) Lis-HC1 (pH 7,
5), 4TIIM Mg C12+ 10mM (N
H4) 2SO4,0,IM KCfl# and 0.1
A reaction solution was prepared with a composition of m Mg-NAD so that the total volume was 400 μl. Add 10 units of E. coli DNA to the reaction solution.
Ligase (manufactured by New Bngland Biolabs) was added, and the mixture was incubated at 11° C. overnight. Additional components were added to the reaction solution to give 40 μM each of dNTP and 0.15 mM β-NΔD, and 5 units of E. coli DNA'J gauze and 7 units of E. coli DNA polymerase I (P-L
Bio-chemicals) and 2 units of E. coli ribonuclease H (PL Biochemicals) and 2 units of E. coli ribonuclease H (PL Biochemicals).
(manufactured by Als) and incubated at 12°C and 25°C for 1 hour each.

In the above reaction, circularization of recombinant DNA including cDNA,
The RNA portion of the RNA-DNA duplex was replaced with DNA, producing a complete double-stranded DNA recombinant plasmid.

Example 3. Selection of recombinant DNA containing chum salmon growth hormone cDNA: Using the recombinant and plasmid obtained in Example 2, E. coli C
600SF3 stocks [Cameron 07 (Cameron):
Proceedings on the National Academy of Sciences
, Sci, ) IJSA.

Yu, 3416 (1975)) by Scott
) et al. [Katsuya Shigesada Cell Engineering, 2,616 (19
83)). Of the approximately 10,000 colonies obtained, 4,800 were fixed on nitrocellulose. 23-28 from the N-terminus of chum salmon growth hormone
The synthetic NA corresponding to the amino acid sequence (3rd base is A or G, 9th base is T or C11
The second one is C or T, the 15th one is C or T, and when combined, it becomes a mixture of 16 synthetic NAs).
Eight bacterial strains that strongly associated with the 2p-labeled probe at 40°C were selected [Grunstein-Hognes
in-Hogness) method, proceeding
Off the National Academy Off Science (Proc, Natl, Acad, Sci, )
USA, 72.3961 (1975): ]. The eight bacterial strains obtained were analyzed by Southern's method [Journal of Molecular Biology (J, Mol.

Biol, ), Tei, 503 (1975) E, the above probe and a synthetic NA probe corresponding to the amino acid sequence near the C-terminus (3rd base is C or T, 6th base is Δ or G, 9
The association was also confirmed with the number A, T, G, or C, and the 12th number G or 8, which when combined results in a mixture of 32 synthetic NAs. These plasmids were named pSG) II, 3.6, 8゜9, and 10.14.17, and all of them have DNA sequences predicted from the amino acid sequence of chum salmon growth hormone, so they are believed to contain growth hormone cDNA. It was thought that there would be.

Example 4. Base sequence of the plasmid pSGH14: The eight plasmids obtained above were digested with various restriction enzymes, and the cleavage map of the cDNA portion was determined. Based on the location of the restriction enzyme site, the obtained plasmids can be classified into three groups: psGl (l, 6.9.10゜17 group, p
They were divided into SGH3 group, pSGH8, and pSGH14 group. The restriction enzyme map of each group is shown in Figure 2.

Among these, the entire base sequence of the plasmids of the psGlll group, especially pSG) II, has already been determined, and the amino acid sequence predicted from the CDNA sequence is similar to that near the N-terminal and The cDNA completely matches the amino acid sequence near the C-terminus, and it has been confirmed that the cDNA encodes Shirojike growth hormone.
and 59-213360).

Furthermore, among the plasmids pSGH8 and ll5G), which have different restriction enzyme cleavage sites from this group, pSG) contains a longer cDNA that is thought to have a full-length cDNA.
114, the entire nucleotide sequence of its translated region was determined using the Sanger method using M13 phage [Sanger (San
ger) et al., Proceedings of the National Academy of Sciences (Proc.

Natl, Acad, Sci,), Mouth SA, 74
, 5463 (1977)
sham) M13 Cloning and Sequencing Handbook (cloning and seque
ncing handbook)]. The sequences are shown in Table 1.

-Genetic Code [: Llniversa
l] 悌1 &MuuAIALy l UAU
I Ijlj^shi ULL^^CTGCACTCTGTA
AArgLysSerLeuGIu^1aAsrlcy
In Table 1, from a comparison with the known growth hormone, it is thought that base numbers 1-66 encode the signal peptide, and base numbers 67-630 code for the mature peptide of the new growth hormone.

The polypeptide encoded by the cDNA is pSG
The 22 amino acid sequences of the signal peptide are completely identical to the polypeptide encoded by II), but the mature peptide portion differs in 12 amino acids out of 188, as shown underlined in Table 1. ing. The N-terminus 4 was also determined from Shijiga-grown Polmonbolipeptide.
Even in the 0 amino acid sequences, 5 amino acids are clearly different, and it is thought that CON^ contained in psGll14 encodes a new growth hormone for fish different from the psHGl group. Escherichia coli containing the recombinant plasmid that has integrated pSGH14 is Bscherichi.
a coli ESGH14 (FERMBP-611)
It was deposited with the Institute of Fine Technology on September 20, 1980.

Example 5. Construction of a recombinant plasmid encoding a new Shiroji moth growth hormone: (1) Recombinant plasmid pSGHII89 encoding mature Shiroji moth growth hormone from pSG)114
Creation: DNA encoding a new chum salmon growth hormone
Plasmid psG8145K containing 20mM)
MCI (pH 7,5), 10mM MgCl1
2, and 100g of a solution containing 10mM NaCl, and restriction enzyme Mbon (New Engl.
and Bio Labs) 1O units, and 37
Digestion reaction was carried out at ℃ for 3 hours. Next, add 5 degrees of NaCl1a.
The solution was adjusted to 9mM, 10 units of pvull was added, and a digestion reaction was performed at 37°C for 3 hours. After this reaction solution was subjected to polyacrylamide gel electrophoresis, a DNA fragment of about 0.05 μm of toabp corresponding to the vicinity of the N-terminus was obtained by the DEAE-paper method.

Next, pSGH145ttg was dissolved in 20mM Tris-HCl.
(p)17.5), 10mM M g C12 and 50+++M NaCj! (hereinafter referred to as “Y-
50 buffer solution (abbreviated as ``buffer solution'')) dissolved in 40 minutes, pvu]
Add 10 units each of I, HindI[I, and heat at 37°C.
A time digestion reaction was performed. The reaction solution was analyzed by LC, T method to obtain approximately 3.3 ml of growth hormone containing the C-terminal side of the growth hormone encoded by pSGH14, the 3'-untranslated region, and a portion of the vector.
Approximately 0.5 Kb DNA fragments were obtained.

On the other hand, in order to add the translation initiation codon ATG necessary for the expression of the DNA encoding mature chum salmon growth hormone, and to connect the vector DNA and the above DNA, the following DNA was added.
An IJ linker was synthesized.

(2) Integration of the region encoding mature chum salmon growth hormone of pSGHn 89 into the expression vector pGELl: Dissolve pSGHIf 89 5■ in 40J11 Y-50 buffer, add 1 each of Ram) II and old ndII [
0 unit was added and the digestion reaction was carried out at 37°C for 3 hours. About 0.1 rhinoceros of about 1200 bp DNA fragment encoding the entire adult chum salmon growth hormone was obtained from the reaction solution by the LC,T method.

Separately, pcεL15■ was dissolved in 404 Y-50 buffer, 10 units each of 13amHI and old ndnI were added.
Digestion reaction was carried out at 37°C for 3 hours. From this reaction solution, LOT
By this method, about 0.1 g of a DNA fragment of about 2.7 b containing a tryptophan promoter was obtained.

psGl obtained above (n old nd■-BamHI of B9
Fragment (approximately 1200 bp) 0.01 g and pGεL old nd nl -BamH [fragment (approximately 2.7 Kb) 0.
015g to 50+nM) Lis-HC1(p)I7.5)
, 10mM MgCj! , , Solution 3 containing 10mM dithiothreitol and lff1M ATP
The mixture was dissolved in 0.0 ml of water, 6 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and a binding reaction was performed at 4° C. for 18 hours.

First, wood-like DNA, 14mer and 9mar were processed using the usual triester method [R, Crea].
LA: Broaching the National Academy of Sciences (Proc. Natl.

^cad, Sci,) LISA,, 75.5765
(1978)). 14mer and 9m
39 pmole each of er single domain DNA at 50 mM
) Lis-HC1 (pH 7,5), 10mM M
g CI22, 10 mM dithiothreitol and 1
Dissolve in 20 scraps of a solution containing mM ATP, add 6 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), and heat at 37°C.
The phosphorylation reaction was carried out for 60 minutes.

Mbo If-Pvu derived from pSGH14 obtained above
I [fragment (103bp) (1, (189mole,
PvuU-Hindlllr fragment (approximately 1200 bp)
0.02 pmoles in 50mM Tris-HCl (p
H7,5>, 10mM MgCl12.1On+M
Solution 30 containing dithiothreitol and 1mM ATP
Dissolve in J11 and add the above synthetic NA! J-oxidation reaction solution 5JJ11 was added. Add T4DNA to this mixture! J
Six units of gauze were added and the binding reaction was carried out at 4°C for 18 hours.

Using the reaction solution, E. coli HBIOI (Polyvar (Bo
Gene, 2, 75(1)
977) strain was transformed to obtain Apl colonies, plasmid DNA was recovered from these colonies, and psGII n 89 shown in Figure 3 was identified as 1st grade. pSGHII B
The structure of 9 is the old ndlI [, Xbal, Bgl U
, BamHI and confirmed by agarose gel electrophoresis.

E. coli strain 88101 was transformed using the reaction solution to obtain A
A colony of plasmid D was obtained from this colony.
NA was collected to obtain pSGHII C2 shown in FIG. The structure of psGII II C2 is εcoRI, H
ind III.

It was cleaved with C1al, Bgl II, and Ram old, and confirmed by agarose gel electrophoresis.

pSGHII B9, pSGHII C2'; r-containing Escherichia coli strain
ESGHII C2FIERM 0P-708
and the same IEsGH] IB9 FBRM BP-707
Both have been deposited at the National Institute of Fine Technology.

Example 6. Production of a new chum salmon growth hormone peptide using E. coli containing pSGHII C2: Using the recombinant plasmid pSGHn C2 obtained in Example 5, E. coli W3]10strA strain (F
BRMBP-732) was transformed. Obtained Apr
Transfer the colony to 8 ml of MCG medium [0.6% Na2) I
PO<, 0.3%KH2PO, 0.5%NaCl1
．． 0.1%NH,CI! , 0.5% glucose, 0
．． 5% Casamino Acids, 1mM ugso4゜4u
/+++l vitamin B l, pH 7,2),
The cells were cultured at 30°C for 18 hours. The obtained culture solution was 8.00
The cells were collected by centrifugation at 0 rpm for 10 minutes. After suspending this bacterial cell in laemmli sample buffer,
DS-polyacrylamide gel electrophoresis was performed and stained with Coomassie brilliant blue, and the molecular weight was approximately 25.0.
A polypeptide band was detected at the 00 site. This band was not present when E. coli not containing the plasmid was used. As a result, it was found that E. coli harboring psGII n C2 produced a large amount of chum salmon growth hormone polypeptide.

Effects of the Invention According to the present invention, a recombinant DNA incorporating a DNA encoding a novel fish growth hormone polypeptide and a microorganism containing the recombinant DNA can be obtained. Can be used for mass production.

[Brief explanation of drawings]

Figures 1 (1) and (2) schematically show the process of cDNA synthesis by the Okayama Haag method and the construction of a recombinant plasmid containing the DNA. FIG. 2 shows restriction enzyme maps of cDNAs contained in pSGH1, pSGH3, and psGI114. Figure 3 shows the recombinant plasmid pSGHII [19, p
The construction process of SGHII C2 is shown. Figure 1 (a)
↓H, 1n4IIIAi conversion Figure 1 (2) Figure 2 C1 piece 1-18) Procedural amendment (voluntary) April 13, 1985

Claims (8)

[Claims] (1) A novel fish growth hormone polypeptide having the peptide sequence shown in Table 1. (2) The growth hormone of fish is herring (Clupeif).
2. The polypeptide of claim 1, which is a novel growth hormone of A. ormes). (3) DNA encoding a novel fish growth hormone polypeptide having the peptide sequence shown in Table 1. (4) A recombinant DNA incorporating DNA encoding a novel fish growth hormone polypeptide having the peptide sequence shown in Table 1. (5) The recombinant DNA of claim 4, named plasmid pSGH14. (6) A microorganism containing a recombinant DNA into which DNA encoding a novel fish growth hormone polypeptide having the peptide sequence shown in Table 1 has been incorporated. (7) The microorganism according to claim 6, wherein the microorganism belongs to Escherichia coli. (8) culturing in a nutrient medium a microorganism containing a recombinant DNA incorporating DNA encoding a novel growth hormone polypeptide for fish having the peptide sequence shown in Table 1;
A method for producing a fish growth hormone polypeptide, which comprises accumulating the fish growth hormone polypeptide in the culture and collecting the polypeptide from the culture.