JPS6115699A - Growth hormone of fish - Google Patents

Abstract

PURPOSE:To obtain growth hormone of fish by genetic engineering method, by synthesizing cDNA from RNA extracted from the pituitary of salmon, determining a base sequence of cDNA depending upon it. CONSTITUTION:Growth hormone peptide of fish having a peptide sequence shown by the table. This peptide is obtained as follows. Namely, mRNA of growth hormone of fish (especially one obtained from the pituitary of salmon) is used as a template to prepare cDNA showing complementation of the mRNA, and a recombinant plasmid integrated with the cDNA is prepared. The recombinant plasmid is further inserted into a host such as Escherichia coli, etc. This transformant is caltivated, to give the aimed growth hormone.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a D'NA encoding a fish growth hormone polypeptide, a recombinant DNA incorporating the DNA, a microorganism containing the recombinant DNA, and a microorganism containing the recombinant DNA. The present invention relates to a method for producing a fish growth hormone polypeptide to be used. 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,
For human growth hormone, U. J. Lewis
La I Koyote J, 8m, Chem, Soc,, 80
.

4429 (1958), A. S. tlartre.
Biochem by e.

J6. Publication 0, 754 (1966), C.tl.
Arch, Biochem, Bio by Li et al.
phys, octa (Suppl,), Yu.

327 (1962).

There have been many reports that fish growth hormones have been isolated, but there are only a few that are reliable in terms of their physiological activity and protein chemical properties. Examples of reliable reporting include:

Example of isolation from tilapia S, W. Farmer et al., Gen.

Con+p, Endocrin, 30.91 (
1976).

Isolation example from sturgeon S, III, Farme
r et al., Endocrinology, 108.377
(1981).

Isolation example from carp A, F, Cook et al., Gen, C
omp.

Bndorcrin, 50.335 (1983).

On the other hand, regarding the growth hormone gene of mammalian animals, the rat growth hormone gene CP, H, Seeburg et al. = Nat
ure 270486 (1977)), the bovine and porcine growth hormone gene CP, H, Seeburg et al.
DNA, 2°37 (1983): ], Human Growth Hormone Gene CJ, A.

Martial et al.: 5science, 205.6
02 (1979):] are already known, but
There are no reports yet on growth hormone genes in fish.

The present inventors previously extracted and purified growth hormone from salmon pituitary gland, and determined the amino acid sequence (31 pieces) from the N-terminus. It has also been confirmed that this substance has a growth-promoting effect on teleost fish [Patent Application No. 591,867]
0).

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 complementary to fish growth hormone polypeptide and producing recombinant DNA and microorganisms containing it, which can be used in the production of fish growth hormone. That is, from the salmon pituitary gland to the metsenger RN.
A (mRNA) is extracted and complementary to this N A
Cloning the salmon growth hormone gene by synthesizing (cDNA), then synthesizing a DNA probe corresponding to the amino acid sequence near the N-terminus of salmon growth hormone, and selecting cDNA that hybridizes with this DNA. succeeded in. Furthermore, the entire base sequence of this cDNA was determined, and the present invention was completed.

The present invention will be explained in detail below.

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

Specifically, fish growth hormone mRNA is used as a template to prepare a DNA (cDNA) complementary to the mRNA, and a recombinant plasmid incorporating the cDNA is prepared.Furthermore, the recombinant plasmid is introduced into a host microorganism. The DNA and the recombinant plasmid can be used to amplify the salmon growth hormone gene, especially in bacteria such as Escherichia coli. Useful for mass production.

Therefore, the present invention provides a DNA encoding a fish growth hormone polypeptide and a recombinant DNA incorporating the DNA.
A microorganism containing A and recombinant DNA is provided.

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 chum salmon, and this was added to oligo dT cellulose (oligo dT cellulose).
e) polyadenylic acid (poIJ) by passing it through a column
RNA having A) (poly A′″ RNA) is isolated.

Using this polyA'' RNA as a template, double-stranded DNA is synthesized using reverse transcriptase.The recombinant is produced by inserting the recombinant DNA into a vector DNA such as E. coli plasmid DNAΔ using in vitro DNA recombination technology. A recombinant plasmid having DNA complementary to chum salmon growth hormone mRNA is selected.

Next, the method for producing the DNA and recombinant plasmid of the present invention will be specifically explained.

The pituitary gland is removed from a captured chum salmon and immediately frozen in liquid nitrogen. This frozen pituitary gland was treated with guanidium inthionanate (guanidiumisoth).
iocyanate), crush, and solubilize. Next, C5Cjl! The solution layer is layered, and after ultracentrifugation, the total cytoplasmic RNA is obtained as a precipitate. Alternatively, only RNA can be precipitated and recovered by adding LiCj2 to the guanidium inthiocyanate solubilized product.

The extracted RNA was NaCj! Or dissolve in a high salt concentration (e.g. 0.5 M) solution of KCj2 and add oligo(dT
) through a column of cellulose containing poly(A)
Adsorb RNA onto the column. Water, 10m M) I
Elute with a low salt solution such as JSu HCβ buffer to isolate the mRNA with poly(A>).

Below, Okayama-Berg's method
a &Berg; J, Mo1. Ce11. Biol
, 2, 161 (1982)), synthesis into cDN and its integration into a vector are performed.

First, vector primers are synthesized. As a vector, for example, pCDVl can be dissolved in a suitable solution, such as Tris-H.
Cl buffer (e.g. pH 7, 5, 10mM), M
g C+22 (e.g. 6mM), NaCl1 (e.g. 10mM) in a solution containing pnI and p
Cut the Kpn 1 site of CDVl.

This DNA was diluted with Tris-H (l buffer (e.g. pH 6,
8,301nM), sodium cacodylate (e.g. 140mM), CaCl2 (e.g. 1mM), dithiothreitol (e.g. O: 1mM) and dTTP.
(for example, 0.25 mM) with terminal deoxynucleotidyl transferase at a constant temperature (for example, 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 added to Tris-HCl buffer (e.g. p) (7, 5, 10mM),
Low melting point agarose gel electrophoresis after cleavage with EcoRI in a solution containing MgC12 (e.g. >6mM), Na (,j! (e.g. 100mM), CLars Wiesla
nder :Analytical Biochemistry
Stry, 98, 305 (1979)], and a fragment of approximately 3.1 kilobases was recovered. The DNA was then diluted with NaCf or K (l) at a high salt concentration (e.g. 0.5 M
) Dissolved in a solution and passed through a poly(dΔ) cellulose column to adsorb only vector primer molecules having poly(T) onto the column. water, 10mM) IJ-HCl
Elute with a low salt solution such as a buffer and remove the poIJ (
Isolate only the vector primer molecule to which T) has been added.

Next, linker DNA is synthesized. For example pLIDNA
in a suitable solution, e.g. Tris-H (1 buffer (e.g. p) + 7.5.10 mM), MgCj! 2 (e.g. 6mM), NaCji! (eg, 50 mM) to cleave the PstI site of pLl. This DNA was replaced with dGT instead of dTTP.
The process is the same as for vector primer synthesis except that P is added, and around 15 oligos d (JJM) are added.

The DNA was dissolved in a suitable solution such as Tris-H (l buffer).
For example, pH 7, 5, 10mM), Mg(J!2 (for example, 5mM).

Hind in a solution containing NaCf1 (e.g. 60mM)
Cut at lll. Approximately 0 in agarose gel electrophoresis
．． A 5 kilobase DNA fragment is fractionated and collected using DEAE paper. Thus, linker DNA is obtained.cDNA synthesis is performed using the poly(A)+RNA, vector primer, and linker DNA obtained as above. Poly(A)” RNA and vector primer DNA were added to Tris-H (l buffer (e.g. pH 8,3,
50m1M), MgC,l! 2 (e.g. 8mM),
K(1! (e.g. 30mM), 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
°C) for a certain period of time (for example, 40 minutes). The 3' end of the RNA-'DNA duplex thus obtained was treated with the same conditions as when a dT strand was added to the vector primer, except that dTTP was changed to dCTP. Oligo d(J
Add around 15 JIs.

This DNA was added to a Tris-HCA buffer (e.g. pH 7,
5, 10 m M ), M g CR2 (e.g. 6
mM).

Hindl in a solution containing NaCl (e.g. 60mM)
Cut with ll. Add the previously prepared linker to this DNA.
Mix the DNA and add Tris-H (l buffer (e.g. pH
7, 5, 20mM), Mg(12 (e.g. 4mM)
, (NH4)2SO4 (e.g. 10mM), K
(1! (e.g. 0.1M), β-nicotinamide adenine dinucleotide (β-NΔD) (e.g. 0.'
1mM) with E. coli DNA ligase for a certain period of time (e.g. 16 hours) at a certain temperature (e.g. 1mM).
Incubate at 2°C. In this way, cDNA and linker DNA are circularized. Add dAT to this reaction solution.
P, dTTP.

Add dGTP and dCTP to a final concentration of 40 μM, add E. coli DNA IJ gauze, E. coli DNA polymerase 11 and E. coli ribonuclease H, and add RN.
By converting part A to DNA, complete double @J
A recombinant plasmid containing fl c DNA is obtained.

Using the thus obtained recombinant plasmid, Escherichia coli, for example, Escherichia coli C600SF8 strain, is grown by the method of 5cott et al. [Katsuya Shigesada: Cell Engineering 1.

6!6 (1983):1. Since the ampicillin resistance gene is present on the recombinant plasmid obtained above, the transformed E. coli exhibits resistance to ampinlin. The following method is used to treat these ampicillin-resistant (A
p') It is generally used to select from bacterial strains a strain carrying a novel recombinant plasmid DNA having a gene complementary to fish growth hormone mRNAΔ. That is, the transformed strain obtained above was immobilized on a nitrocellulose filter, and a synthetic NA having a D/NA sequence predicted from the known amino acid sequence of chum salmon growth hormone was immobilized on a nitrocellulose filter.
associate with the probe and select those that associate strongly (G
runstein-Hogness method, Proc.
, Natl.

Acad, Sc1,, IJsA,, 72.396
．． 1 (1975)). Probe DNA was prepared using the usual triester method [J, Am, [hem, Soc,.

Machi, 7327 (1975)). Synthetic N
Selection using the A probe was performed using the method of 5outhern et al. [J
, Mo1゜Biol, 98.503 (1975)
), and by this method, recombinant plasmid DNA having a gene complementary to chum salmon growth hormone mRNA can be identified.

The novel recombinant plasmids of the present invention can be used for large scale production of fish growth hormone polypeptides by microorganisms such as E. coli or eukaryotic cells.

Examples of the present invention are shown below.

Example 1. Po'JA+RNA from chum salmon pituitary gland
Preparation: Guanidium thiocyanate-cesium chloride method from chum salmon pituitary gland [edited by Maniatis et al.

Molecular Clotting, p19
6. Published by Cold Spring Harbor;
Katsuya Shigesada, Cell Engineering, 2, 616 (1983):
RNA having polyA was prepared as follows according to 1.

, 2g frozen pituitary gland of chum salmon (approx. 30 individuals) 4M
Guanidium thiocyanate, 0.5% sarcosine, 5
mM sodium citrate (pH 7) and 0. IMβ-
The mixture was crushed and solubilized using a Teflon homogenizer (5 rpm) in 10 ml of a solution consisting of mercaptoethanol. Pass this homogenate through an 18G needle several times to extract the DNA.
was divided. 5.7MCs'C, CO, IM, ED
12 ml of each TA (pH 8) solution was dispensed into ultracentrifuge tubes, and the homogenate was layered thereon. old tachi
RP S 4.0 35,000 rpm at low speed
After centrifugation for 15 hours, RNA was collected as a precipitate. R
Precipitate the NA in Tris-H containing 1mM EDTA (1
(pH 8,0) solution 10ml 1k: Dissolution, phenol-
After extraction with chloroform, it was recovered by ethanol precipitation. , about 1 mg of the obtained RNA was added to 10 mM) Lis-HCf
1 (pH 8,0) and 1mM EDTA.

Incubate at 65°C for 5 minutes and add 0.1 ml of 5MN.
aCl1 was added. The mixture was chromatographed on an oligo dT cellulose column (PL Biochemicals). m with adsorbed polyA
The RNA was eluted with a solution consisting of 10 mM) Lis-HCβ (pH 8.0 and 1 mM EDTA) to obtain about 10 μg of polyA-containing mRNA.

Example 2. cDNA synthesis and insertion of the DNA into a vector; Okayama-Berg method CJ, Mo1. Ce
1l, Biol, r 2.161 (1982)), synthesis of cDNA and construction of a recombinant plasmid incorporating it were performed. An outline of the process is shown in FIG.

pCDV l [Okayama & Japan e
rg: J, Mol, Ce1l.

Biol, 3.280 (1983) ] 400. L
Eg 1QmM) Lis-H (1 (pH 7,5), 6mM
MgCL and 10mM NaCj! In addition to 300 uj2 of a solution consisting of 500 units of Kpnl (
(manufactured by Takara Shuzo Co., Ltd.) and reacted at 37°C for 6 hours, and the plasmid was cleaved at the Kpn 1 site. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation. Approximately 200 μg of the Kpn I-cleaved DNA was added to 4Q
mM sodium cacodylate, 30 mM Tris-HCR (
pH 6,8), 1mM CaCjl! 2 and 0.1
Add dTTP to a buffer solution (hereinafter abbreviated as TdT buffer) consisting of mM dithiothreitol (hereinafter abbreviated as DTT).
was added to 200 μl of a solution containing 0.25 mM, and further 81 units of terminal deokine nucleotidyl transferase (hereinafter abbreviated as TdT) (P
-L Bioche-micals) and 3
The reaction was carried out for 11 minutes at 7°C. Here, Kpn of pCDVl
Approximately 67 po1JdT chains were added to the 3' end of the I cleavage site. From this solution, phenol-chloroform extraction and ethanol precipitation were carried out to obtain pCDV to which po-1JdT was added.
Approximately 100 μg of I DNA was recovered. The DNA is 1
0mM) Lis~H(1 (pH 7,5), 6mM Mg
Cβ2゜Buffer consisting of 100mM NaCl 150
In addition to μβ, 360 units of EcoRI (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was reacted at 37° C. for 2 hours. After the reaction product was subjected to low melting point agarose gel electrophoresis, approximately 3. IKb DNA
The fragments were collected and about 60 μg of po1JdTIJl-added pC
Got DV1. The DNA was diluted with 10mM) Lis-HCβ (
Solution 5 consisting of pH 8,0) and 1mM EDTA
After incubating at 65° C. for 5 minutes, the mixture was cooled on ice and 50 μl of 5M NaCl was added. The mixture was subjected to chromatography on an oligo dA cellulose column (manufactured by Collaborative Research). Po1. Those with sufficient ldT binding were adsorbed onto the column and eluted with a solution consisting of 10mM) Lis-HC1) (pH 8,0) and 1mM EDTA.
27 μg of 1 (hereinafter abbreviated as vector primer) was obtained.

Next, linker DNA is prepared.

p L 1 (Okayama & Berg:
J, Mol, Ce11. Biol.

3, 280 (1983) E approx. ILug at 10 mM) Lis-HCj! (pH 7,5), ・6mM Mg(J
'2 and 50mM NaCj! A buffer solution consisting of 20
In addition to 0 μl, 50 units of PstI (manufactured by Takara Shuzo Co., Ltd.) was added and reacted at 37°C for 4 hours to remove PstI in pLIDNA.
It was cut at the stI 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 μβ of a solution containing P, T d T (P
-L Biochemicals) was added and incubated at 37°C for 13 minutes to increase the Ps of pLl.
Approximately 14 dG molecules were added to the 3' end of the tI cleavage site.

D by ethanol precipitation after phenol-chloroform extraction
NA was collected. 100 μj of the DNA! 10mM)
1. -HCj! (pH 7,5), 6mM MgC
100 μl of buffer consisting of L and 5QmM Na'Cβ
In addition, 80 units of HindIII (manufactured by Takara Shuzo Co., Ltd.) was added and incubated at 37°C for 3 hours.
'A) was cut at the lind111 site. The reaction product was fractionated by agarose gel electrophoresis, and a DNA fragment of approximately 0.5 Kb was separated using the DEAE paper method [Dretzen et al., Anal.

Biochem, 112.295 (1981)
:], and oligo d (linker-DN with JJI
A (hereinafter simply referred to as linker DNA) was obtained.

Approximately 2 μg of po'J'(A) RNA prepared above and approximately 1.4 μg of vector primer were mixed with 50 mM Tris-HCl (
pH 8,3), 8mM MgCj72.30mM
KCjl! , 0.3mM DTT, 2mM dNTPs
(dATP, dTTP, dGTP and dCTP) and 10 units of glue bonuclease inhibitor (P-L
Solution 22 consisting of (manufactured by Biochemicals)
．． 3μβ, add 10 units of reverse transcriptase (manufactured by Seikagaku Corporation), incubate at 37°C for 40 minutes, and mR
Complementary NA was synthesized. The reaction product was extracted with phenol-chloroform and precipitated with ethanol.
Vector primer DN with double NA-DNA double layer added
A was collected. The DNA was treated with 66 μM dCTP and 0.
Dissolved in 20 μm of TdT buffer containing 2 μg of polyA,
4 units of T d T (PL Biochemical
als) and incubated at 37°C for 8 minutes.
Twelve dC chains were added to the 3' end of c[)NΔ. The reaction product was extracted with phenol-chloroform, and the cDNA-vector primer DNA to which the dC compound had been added was recovered by ethanol precipitation. The DNA was purified by Tris-H (1!
pH 7,5>, 6mM MgCj22 and 60mM
A solution consisting of NaCA (40% dissolved in DzA, 20%
Add the units of Hl and ndlIl (manufactured by Takara Shuzo Co., Ltd.) and make 37
C. for 2 hours and cut at the HindllI site. The reaction product was extracted with phenol-chloroform and precipitated with ethanol to obtain 0.5 pmole of dC-adducted cDNA.
A-vector primer DNA was obtained. The DNA0.0
8 pmole and the linker-DNA 0.16 above
pmole at 40 μA Tris-HC,A(p)(7,
5), 0. IM NaCj7 and 1mM E,D
Add to 40 μl of solution consisting of TA, 65°C, 42°C, 0
℃ for 10 and 25 minutes, respectively.

Incubated for 30 minutes. 20mM) Squirrel-HFJ
! (pH 7,5), 4mM MgC, l'2.10
Composition of mM (NH4)2SO4゜0, IM KCffl and 0.1 mM β-NAD, total amount 400μβ
A reaction solution was prepared as follows.

Add 10 units of E. coli DNAIJ gauze (New
(manufactured by England Biolabs) and 11
- Incubated overnight. The reaction solution was diluted with 40 μM of d
Add components to NTP, 0.15mM β-NAD, and add 5 units of E. coli DNA! J gauze, 7 units of E. coli DNA polymerase I (PL Bio-chem
icals) and 2 units of Escherichia coli ribonuclease H (P-L Biochemicals) were added and incubated at 12°C and 25°C for 1 hour each.

In the above reaction, the recombinant DNA containing cD'NA was circularized and the RNA-DNA duplex material NA portion 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 plasmid obtained in Example 2, E. coli c6
00sP8 strain [Cameron: Proc, Natl
, Acad, Sci.

USA, Yu, 3416 (1975)] 5cott
[Katsuya Shigesada: Cell Engineering, 2,616 (198
3) Transformation was performed according to ). Of the approximately 10,000 colonies obtained, 4,800 were fixed on nitrocellulose. Synthetic NA corresponding to the 22nd to 27th amino acid sequence from the N-terminus of chum salmon growth hormone, i.e. (3
The 115th base is A or G, the 6th base is Δ or G, the 12th base is T or C, and the 115th base is C or T, resulting in a mixture of 16 synthetic NAs).
We selected eight bacterial strains that strongly associated with the probe labeled at 40°C [Grunstein-Hogness method,
Proc, Natl, Acad, Sc1, LIS
A.

72.396'1 (1975)). The 5outhern method [J, Mo1. Biol
, 98.503 (1975)), the above probe and the synthesis corresponding to the amino acid sequence near the C-terminus, D
NA probe (3rd base is C or T, 6th base is A or G, 9th base is either A, T, G, or C, 1
The second one is G or A, which can be combined to form a mixture of 32 synthetic NAs). These plasmids are psGHl, 3, 6, 8°9, 10
．． 14.17, and both had DNA sequences predicted from the amino acid sequence of chum salmon growth hormone, so they were thought to contain growth hormone cDNA.

Example 4. Base sequence of the plasmid pSGII 1: 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: psGtll, 6.9.10゜17 group, psGtll, 6.9.10゜17 group,
They were divided into sGH3 group, PSGH8 and 14 groups. The restriction enzyme map of each group is shown in Figure 2.

Next, we analyzed the entire nucleotide sequence of the translated region of the plasmid, especially pSGH1, which shows the strongest association with the synthetic NA probe conducted in Example 3 and is thought to contain almost full-length cDN8. M137
Sanger method using y-shi [Sanger et al., Pr.
oc, Natl, 8cad, Sct, LISA
, 74.5463 (1977): Mersham
Company M l 3 cloning and seque
ncinghandbook). The sequences are shown in Table 1. In Table 1, base numbers 1-66 code for the gunnar peptide, and base numbers 67-630 code for the mature peptide of chum salmon growth hormone. The amino acid sequence predicted from the cDNA sequence contained in pSGII 1 completely matches the amino acid sequence near the N-terminus and near the C-terminus determined from chum salmon growth hormone peptide, and
It was confirmed that NA encodes chum salmon growth hormone. psG January, PSGH3.

E. coli containing pSGH8 (BSGH1, BSG, respectively)
H3゜ESCH8) is dated June 23, 1982, and is posted on FB
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as RM BP-551°552 and 553.

-Genetic Code CLlniver5a
To A 1 Table Argl-ysSerLeu
Effects of the Invention on GIu According to the present invention, microorganisms containing recombinant DNA and recombinant DNA incorporating DNA encoding a fish growth hormone polypeptide can be obtained, and these microorganisms can improve the growth of fish. It can be used for mass production of hormone polypeptides.

[Brief explanation of the drawing]

FIG. 1 schematically shows the process of cDNA synthesis by the Okayama-Berg method and the construction of a recombinant plasmid containing the DNA. FIG. 2 shows restriction enzyme maps to cDNs contained in pSGH1, pSGH3, and psGH8. ↓Oka coRI Jikado U
J) (IRDL [SM I and 1 Figure Vector Brine -DNA -Swordsmith ===== Type PTTTT ηRNA 8 ＾ He----------------------------------------------------------------- g month 17th

Claims (11)

[Claims] (1) A fish growth hormone polypeptide having the peptide sequence shown in Table 1. (2) The growth hormone of fish is herring (Clupeif).
Claim 1, which is a growth hormone of
term polypeptide. (3) D encoding a fish growth hormone polypeptide
N.A. (4) The DNA of claim 3, in which the fish growth hormone polypeptide has the peptide sequence shown in Table 1.
. (5) D encoding a fish growth hormone polypeptide
Recombinant DNA incorporating NA. (6) The recombinant DNA of claim 5, wherein the fish growth hormone polypeptide has the peptide sequence shown in Table 1. (7) Plasmid pSGH1, pSGH3 or pSG
Recombinant DNA of claim 5 named H8
. (8) D encoding a fish growth hormone polypeptide
A microorganism containing recombinant DNA incorporating NA. (9) The microorganism according to claim 8, wherein the fish growth hormone polypeptide has the peptide sequence shown in FIG. (10) The microorganism according to claim 8, wherein the microorganism belongs to Escherichia coli. (11) Cultivating a microorganism containing a recombinant DNA incorporating DNA encoding a fish growth hormone polypeptide in a nutrient medium, accumulating the fish growth hormone polypeptide in the culture, and extracting the fish growth hormone polypeptide from the culture. A method for producing a fish growth hormone polypeptide, which comprises collecting the polypeptide.