JPS62230799A - Growth hormone gene of fish and polypeptide coding said gene - Google Patents

Abstract

NEW MATERIAL:The growth hormone polypeptide of fish having the peptide sequence of formula. USE:Growth-promoting agent for fish. PREPARATION:Whole DNA is separated from pituitary gland of a fish belonging to Apodes order, Actinopterigii class (e.g. eel) and is passed through an oligo-dT- cellulose column to separate RNA containing polyadenylic acid. A cDNA is produced by a reverse transcriptase using the RNA as a template and is integrated into a vector DNA such as a plasmid DNA of a host microorganism such as E.coli. The vector DNA is inserted into a host microorganism to effect the transformation of the microorganism. The transformed host microorganism is cultured in a medium to accumulate the growth hormone polypeptide of fish in the culture product. The objective polypeptide of formula can be separated from the culture product.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a DNA encoding a fish growth hormone polypeptide, a recombinant DNA incorporating the DNA, a microorganism containing the recombinant DNA, and a fish using the microorganism. The present invention relates to a method for producing a 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,
Regarding human growth hormone, the journal
of American Chemical Society 4 (J,A
m, Chem, Sac, ), 80, 4429 (1
958), A.1.Bathley (A.S.Ha.
Biochemical Journal (B
iochem, J.).

■Archives of Biochemistry and Biophysics (Archives of Biochemistry and Biophysics) (Arch, Biochem, Biophys,
(Suppl, )), Yu, 327 (1962).

Regarding fish growth hormone, the following are examples of isolated reports.

Isolation example from tilapia: Niss W. Farmer et al., General Ant.
Comparative Endocrinology (Gen.

Comp, Endocrin, ), 30.9H197
6).

Isolation example from sturgeon: Nis d'abrini pharma (
S., W. Farmer et al., Endocrinology, 108. 37
7 (1981).

Isolation example from carp: A.F. Tac (A, F.

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

50, 335 (1983).

Isolation example from chum salmon; JP-A-60-214798 On the other hand, regarding the growth hormone gene of free-breeding mammals, the rat growth hormone gene [B.H. Seabarb (P, H, S
eeburg et al.: Nature 2
70486 (1977)), bovine and porcine growth hormone genes [P.H.
ee-burg) et al.: DFA (DNA)
, 2.37 (1983)), human growth hormone gene [J.A. Marti
al) et al.: Science, 205.
602 (1979)) are already known, and among the fish growth hormone genes, the chum salmon growth hormone gene has already been isolated by the present inventors (Japanese Patent Application Laid-Open No. 15699/1983).

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,
Collection of NA complementary to fish growth hormone polypeptide, recombinant DNA containing the same, and the recombinant DNA that can be used for the production of fish growth hormone
We succeeded in producing microorganisms containing A. Specifically, Metsenger RNA (mRNA) is extracted from the eel pituitary gland, cDNA complementary to this is synthesized, and then a DNA probe corresponding to the amino acid sequence near the N-terminus of eel growth hormone is synthesized. By selecting a cDNA that hybridized with this DNA, the eel growth hormone gene was cloned, and the entire base sequence of the cDNA was determined. The present inventors conducted further research and found that by culturing microorganisms containing recombinant DNA that had incorporated DNA encoding eel growth hormone, a significant amount of eel growth hormone polypeptide accumulated in the culture. This finding led to the completion of the present invention.

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.

That is, using fish growth hormone mRNA as a template, DNA (cDNA) complementary to the mRNA is prepared, and a recombinant plasmid incorporating the cDNA is prepared. Furthermore, the recombinant plasmid is inserted into a host microorganism. The DNA and recombinant plasmids can be used for amplification of the eel growth hormone gene, especially in bacteria such as Escherichia coli. A microorganism having the recombinant plasmid is useful for producing eel growth hormone in large quantities at low cost.

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

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

Total RNA was prepared from the eel pituitary gland, and this was used as oligo d.
T cellulose (oligo dT cellulose)
) RN with polyadenylic acid by passing through the column
A [poly(A) RNA] is separated. This poly(A)R
Double-stranded DNA is synthesized using reverse transcriptase using NA as a template. Recombinants are obtained 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 DNA and recombinant plasmid of the present invention will be specifically explained.

The pituitary gland is removed from the captured eel and immediately frozen in liquid nitrogen. Guanidine thiocyanate is added to the frozen pituitary gland to crush it and solubilize it. Then add Li(l) and after centrifugation, obtain the total cytoplasmic RNA as a precipitate.

Alternatively, a solubilized guanidine thiocyanate can be layered on the CsC1 solution layer, and after ultracentrifugation, RNA can be obtained as a precipitate.

The extracted RNA was treated with NaCR or KCI high salt concentration (
For example, mRNA containing poly(A) is dissolved in a 0.5M) solution and passed through an oligo(dT) cellulose column.
is adsorbed onto the column. Elute using a low salt solution such as water, 10mM)
A> is isolated.

Below, Okayama-Berg (Okayama-Berg)
) method [Okayama and Barb (Okayama
&Berg); Molecular & Cellular
Biology (Mo1.Ce11.Bial, ) 2
, 16H1982)), synthesis of 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-
1((1! Buffer (for example pt(7,5,1(1m!
J), MgCf2 (e.g. 6mM), NaC
j! (eg, IOmM) to cleave the Kpn I site of pcDVl.

This DNA was added to a Tris-HCl buffer (e.g. pH 6,
8,30mM), sodium cacodylate (e.g. 140mM), CoC12 (e.g. 1mM
M), dithiothreitol (e.g. 0.1mM) and dTTP (e.g. 0.25mM) with terminal deoxy, synucleotidyl transferase at a constant temperature (e.g. 37°C) for a specified time (e.g. 20
Approximately 60 thymidyl residues are added to both 3' ends of the vector DNA. Furthermore, this D
NA was added to Tris-HCl buffer (e.g. pH 7, 5, 1
0mM), MgCl1z (e.g. 6mM), NaCj
! (e.g. 1(11) mM) in a solution containing EcoR
After cutting with I, low melting point agarose gel electrophoresis [Lars Wieslander]
, Analytical High-Off Chemistry (^naly)
tical Biochemistry) 98.305
(1979), hereinafter referred to as the LGT method], and a fragment of approximately 3.1 kilobases is recovered. Then, the DNA was converted into NaCj! Or KCj! high salt concentration (e.g. 0,5
M) Dissolve in a solution and pass through a poly(dΔ) cellulose column. Only vector brimer molecules having J (T) are adsorbed onto the column. Water, 10mM Tris-H'
Elute with a low salt solution such as Cβ buffer and
Only the J(T)-added vector primer molecules are isolated.

Next, linker DNA is synthesized. For example pLI DN
Treat A with Pstl in a suitable solution, such as a solution containing Tris-HCj2 buffer (e.g. pH 7,5, 1On+IJ), MgCL (e.g. 6mM), NaCl (e.g. 50ml) to cleave the PstI site of pLl. . This DNA is used as dGTP instead of dTTP.
The procedure is the same as for vector primer synthesis except that about 15 oligo (dG) chains are added. The DNA is dissolved in a suitable solution such as Tris-HCl buffer (e.g. pH 7, 5, 10mM>, MgCR).
2 (e.g. 6mM).

Hin in a solution containing NaCj' (e.g. 60mM)
Cut at dI[I. DNA fragments of approximately 0.5 kilobases are fractionated by agarose gel electrophoresis and recovered using DEAE vapor. In this way, linker DNA is obtained.

cDNA synthesis is performed using the po'J (A) RNA, vector primer, and linker DNA obtained as described above. Poly(A) RNA, vector primer DNA
A in Tris-HCl buffer (e.g. pt-+8.3.
>50mM, MgCL (e.g. 3mM).

KCj! (e.g. 30mM), dithiothreitol (e.g. >0.3mM), dATP, dTTP.

Reverse transcriptase is reacted in a solution containing dCTP, dC, and TP (for example, 2 mM each) at a constant temperature (for example, 37° C.) for a certain period of time (for example, 40 minutes).

At the 3' end of the RNA-DNA duplex thus obtained, dT
The vector primer except that TP is changed to dCTP (
Oligo (dC) was added under the same conditions as when dT) was added.
Add around 15 chains. This DNA was converted into Tris-H (l
Buffer (e.g. pH 7, 5, 10mM), MgCL (
For example, 6 m M>, N a C1 (for example, 6
Cleave with HindIIr in a solution containing >0mM. This DNA is mixed with the previously prepared linker DNA, and added with Tris-HCj2 buffer (e.g. pH 7, 5, 20mM).
, MgCL (e.g. 4mM>, (NH4)2SO
4 (e.g. 10mM).

HCl (e.g. 0.1 M), β-nicotinamide adenine dinucleotide (β-NAD) (e.g. 0.1
Incubate with E. coli DNA'J gauze for a certain period of time (for example, 16 hours) at a constant temperature (for example, 12 °C) in a solution containing (mM). In this way, cDNA and linker DNA are circularized. dA to this reaction solution
TP, dTTP, dC, TP.

dCTP was added to a g concentration of 40 μM, and E. coli DNA'J gauze, E. coli 08 polymerase ■, and E. coli ribonuclease H were added to convert the RNA portion into DNA, resulting in a recombinant product containing a complete double-stranded cDNA. Obtain the plasmid.

Using the recombinant plasmid obtained in this way, Escherichia coli, for example, Escherichia coli C6(11) SF8 strain, for example, Scots) (
Scott et al.'s method [Katsuya Shigesada: Cell Engineering 2.61
6 (1983)).

Since the ampicillin resistance gene is present on the recombinant plasmid obtained above, the transformed E. coli exhibits ampicillin resistance. The following method uses these ampicillin-resistant (Ap') strains to generate a new recombinant plasmid DNA containing a gene 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 a synthetic N having a DNA sequence predicted from the known amino acid sequence of eel growth hormone was immobilized.
Select the one that associates strongly with the A probe [
Gruns-tein
Hogness) method, Proceedings φ of the National Academy of Sciences (Pro
c, Natl, Acad, Sci,), USA,...
72.3961 (1975)). probe dna
is the usual triester method [Journal of American Chemical Society (J, Am, Chem,
Soc, ), 97.7327 (1975)]. Selection using synthetic NA probes was performed using the method of Southern et al. [Journal of
Molecular Biology (J, Mol.

Biol, ), Sugi, 503 (1975)), it was further confirmed that eel growth hormone m
Recombinant plasmid DNA having genes complementary to RNA can be identified.

An example of a recombinant plasmid obtained in this way is p
It is EGH15. This plasmid can be used as a source of DNA encoding eel growth hormone.

There are two types of eel growth hormone, GH-1 and GH-H. Comparing the amino acid sequences of both, GH-I
[3 amino acid residues are missing from the N-terminus of GH-I (Japanese Patent Application No. 61-151847).

pEf:, H15 obtained above is a plasmid containing DNA encoding GH-1. Therefore, pEGH
It is also possible to excise the DNA encoding GH-1 from 15 and use recombinant DNA techniques to produce a plasmid containing the DNA encoding C,H-n.

The eel growth hormone polypeptide is obtained by excising the DNA from a plasmid containing the DNA encoding eel growth hormone, incorporating it into vector DNA, introducing the resulting recombinant DNA into a microorganism, and culturing the resulting transformant. Eel growth hormone polypeptide can be produced by producing and accumulating in culture and collecting it.

A preferred example of a plasmid containing DNA encoding eel growth hormone is the above pE(1,H2S).

Any vector DNA can be used as long as the inserted DNA can be expressed in microorganisms. Preferably, a suitable promoter,
For example, tryptophan (trp), lactose (I
ac) system, PL system, etc., DNA can be inserted downstream, and the internal Shine-Dalgarno sequence (hereinafter abbreviated as SD sequence) and translation initiation codon (
ATG> and the vector DNA is adjusted to an appropriate distance, for example, 6 to 18 base pairs. A specifically suitable vector DNA is plasmid pGL1.
I can give you 11. pGLMl is the plasmid shown in Figure 3, and the E. coli containing it is [! 5cheri
-chiacoli EGLMI (FERM BP
-823) on July 2, 1985, and was deposited with the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology. Recombination between the polypeptide-encoding DNA and vector DNA is achieved by digesting both DNAs with restriction enzymes and then using T4D.
NA! General recombinant DNA ligated using J gauze
This can be done using a method.

In the case of pEGH15 and pGLM1 shown as specific examples, construction is performed as follows. That is, a BanII-BamHI digested fragment containing a cDNA part encoding the eel growth hormone mature peptide and a part of the vector was obtained from pEGH5, and a PL promoter and Cl85 were obtained from pGLM1.
BamHI-BanII [digested fragment containing 7 genes is obtained. On the other hand, a synthetic NA IJ linker as shown below is prepared.

Both of the above DNA fragments and the synthetic NAIJ anchor were combined into T4DN.
A! The recombinant plasmid pUPΔ1 shown in FIG. 3 is obtained by ligating with J gauze. This plasmid has a region encoding mature eel growth hormone (GH-I) linked downstream of the P 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 2-2(11)mM (preferably 10-2(11)mM)
40mM) Tris-HCl (pH 6.0-9.5 preferably pH 7.0-8.0), 0-2(11)mM
of NaCl, 2-30mM (preferably 5-10mM
) in a reaction solution containing MgCL of 0.1-1 (
11) units (preferably 1 to 3 per 1 μg of DNA)
unit) at 20 to 70°C (the optimal temperature varies depending on the restriction enzyme used) for 15 minutes to 24 hours.

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 DNA fragments generated by restriction enzyme digestion was performed using LG
This is performed using the T method, polyacrylamide gel electrophoresis, or the like.

The binding reaction of the DNA fragments is carried out using 2 to 2 (11) mM (preferably 1 (1 to 40 mM)) of Tris-H (1 (pH 6, 1
-9.5, preferably pH7.0-8.0), 2-2
0mM (preferably 5-10mM) MgCL, 0.
1-10mM (preferably 0.5-2,0mM
) of ATP, 1-50mM (preferably 5-10mM
) in a reaction solution containing dithiothreitol.
Using 0.3 to 10 units of ligase at 1 to 37°C (preferably 3 to 20°C) for 15 minutes to 72 hours (preferably 2 hours).
~20 hours).

The recombinant plasmid DNA produced by the ligation reaction is
If necessary, the transformation method of Coher+ et al. [S, N. Cohen et al.: Proceedings of the National Academy of Science
(Proc, Natl, ^cad, Sci,
).

USA, Deaf, 2110 (1972)) into E. coli.

Isolation of recombinant plasmid DNA from Escherichia coli carrying the DNA can be carried out using cesium chloride-ethidium bromide density gradient ultracentrifugation [D.
, Clewe l I) et al.: Proceed 4 Fugu of the National Academy of Science x (
Proc, Natl, Acad, Sci,),
USA.

62, 1159 (1969)) or Birnboim (
[H0C9 Birnboim et al.: Nucleic Acid Research (Nucleic Acids R)
es, ) Yu, 1513 (1979) E, 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 [Proceedings of the National Academy of Sciences
Natl, ^cad, Sci, ),
74. 560 (1977)) or the Sanger method using M13 phage [Sanger (S
Anger) et al.: Proceedings of the National Academy of Sciences (Proc.
Natl, ^cad, Sci, ).

USA, 74.5463 (1977); Mersham M13 Cloning and Sea Fencing, Hafd.
and sequencing hand-book
) ) to be determined.

The eel growth hormone polypeptide of the present invention can be produced as follows.

That is, E. coli is transformed with -12c6 (11) using a plasmid (for example, pflPAI), and E. coli containing pLIPAl is selected from ampicillin-resistant colonies. By culturing E. coli containing pUPA 1 in a medium, eel 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 eel growth hormone polypeptide.

Carbon sources include glucose and fructose.

Lactose, glycerol, mannitol, sorbitol, etc. are nitrogen sources such as NH, (1° (NH=)2
SO4, Casamino Acids, Yeast Extract, Polypeptone, Meat Extract, Bactodryptone, Corn Steep Liquor, etc. Other nutritional sources include K2HPO, KH
2PO4, NaC1゜MgSO4, vitamin B+,
MgCC 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.

Eel growth hormone polypeptide accumulates in the cultured cells after 5 to 90 hours of culture, so collect the cells from the culture.
After treating the bacterial cells with lysozyme, the bacterial cells are crushed by repeated freezing and thawing, and polypeptides are collected from the supernatant obtained by centrifugation according to a conventional polypeptide extraction method.

In addition, the detection of the polypeptide can be done by directly using Laemmli (L) cultured bacterial cells.
aemmli) sample buffer [Laemmli
mml i), Nature + - (Nature), 2
27, 680 (1970)), 5D
S-polyacrylamide gel [Laemm'J
li) method 2 (ibid.) and Western plotting [Towbin et al.
), USA, 76, 4350 (1979)
), followed by enzyme-antibody staining using a secondary antibody labeled with horseradish peroxidase (manufactured by DAKO) [Tabe Fumi: Cell Engineering, 2゜1061 (1983)
).

Examples of the present invention are shown below.

Example 1. Po from the eel pituitary gland! J(A)RN
Preparation of A Poly(A)-containing RNA was prepared from the eel pituitary gland according to the guanidine thiocyanate-lithium chloride method [Cathala et al., DNA, 2.329 (1983)] as follows.

0.5g of frozen pituitary gland of eel (approximately 1(11)0 individuals) was mixed with 5M guanidine thiocyanate and 10mM EDT.
A.

50mM Trix-HCII (pH 7) and 8% (
V/V) 10 ml of solution consisting of β-mercaptoethanol
The mixture was crushed and solubilized using a Teflon homogenizer (5 rpm) in 100 ml of water. Transfer this lysate to a centrifuge tube and add 4M L
After adding 7Qml' of iCβ solution and stirring, the mixture was left standing for 4t20 hours. 9. At the old tachi RP R10C'-ter. (11) After centrifugation at 0 rpm for 90 minutes,
RNA was collected as a precipitate. The RNA precipitate was suspended in 10 Qml of a solution consisting of 4M urea and 2M lithium chloride, and purified with a Hitachi RP R10 rotor.
．． (11) After centrifugation in Orpm for 60 minutes, RNA was recovered as a precipitate again. Precipitate the RNA with 0.1% sodium lauryl sulfate, 1mM EDTA, 10mM)
HCjl! (pH 7.5), extracted with phenol-chloroform, and recovered by ethanol precipitation. Approximately 1 mg of the obtained RNA was
0mM):x-HCfl (pH 8,0> and 1 m
It was dissolved in a solution 11'Ill consisting of lJEDTA. Incubate at 65°C for 5 minutes, 0.1ml
of 5M NaCl was added. Oligo(dT) mixture
Cellulose column [BL Biochemical (P
-LBio-chemical) chromatography (column volume: 0.5 ml). Adsorbed po+
mRNA with J (A) lOmM) Lis-HC1 (
Elute with a solution consisting of pH 7.5> and 1mM EDTA, and pop! Approximately seven rhinoceros mRNAs having I (A) were obtained.

Example 2. CDNA synthesis and insertion of the DNA into a vector: Okayama-Berg's method [Molecular and Cellular Biology]
(Mo1.Ce11.Biol,), 2,161 (19
cDNA was synthesized and a recombinant plasmid incorporating it was constructed according to 82)). An outline of the process is shown in FIG.

pcDVI C Okayama and Barb
-ma & Berg): Molecular and Cellular Biology (Mol, Ce1l, Bi
ol, ), 3.280 (1983) ] 4 (11
) pg 10mM) Lis-HC1 (pH 7,5),
3 (11) μl of a solution consisting of 6mM VIgCL and 10mM NaCl plus an additional 5 (11) units of K.
Add pnI (manufactured by Takara Shuzo Co., Ltd.; unless otherwise specified, all restriction enzymes are manufactured by Takara Shuzo Co., Ltd.), and react at 37°C for 6 hours.
It was cut at the Kpn■ site in the plasmid. Phenol-
After chloroform extraction, DNA was recovered by ethanol precipitation.

Approximately 2 (11) μg of the Kpn I-cleaved DNA was added to 40 m
M Sodium cacodylate, 30mM) Lis-HC1 (p
H6,8), lmM CaCj! 2 and 0.1
Add dTTP to a buffer solution (hereinafter abbreviated as TdT buffer) consisting of mM dithiothreitol (hereinafter abbreviated as DTT).
Add to 2 (11) μl of a solution containing 0.25 mM, and add 81 units of terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT).
(manufactured by P-L Biochemicals),
The reaction was carried out at 37°C for 11 minutes. Here, pCD V,
Poly(dT) at the 3' end of the Kl)TI I cleavage site
Approximately 67 chains were added. Approximately 1 (11) μg of pcDVI DNA to which poly(dT) chains were added was recovered from the solution by phenol-chloroform extraction and ethanol precipitation. The DNA is 1QmM) Lis-HCj! (pH7,
5), 6mM MgCj22,1(11)mM N
aCj! Add to 150 μl of buffer consisting of
0 units of EcoRI was added and the mixture was allowed to react at 37°C for 2 hours. After treating the reactant with the LGT method, about 3. The I K b DNA fragment was recovered, and about 60 μg of po IJ (dT) chain-added pCDV1 was obtained. The DNA was 1QmM) Lis-HC
l! (pH 8,0) and 1mM EDTA (pH 8,0) and 1mM EDTA. added.

The mixture was subjected to oligo(dA) cellulose column chromatography (manufactured by Collaborative Research). Poly(dT) with sufficient chain length is adsorbed onto the column, and this is mixed with 10mM) Lis-HCβ (pH 8,0) and 1mM
Elute with a solution consisting of EDTA,
27 μg of chain-added pcDVl (hereinafter abbreviated as vector primer) was obtained.

Next, linker DNA was prepared.

pLl (Okayama and Barb)
& Berg): Molecular and Cellular Biology (Mol, Ce1l, Biol,),
3.280 (1983)) Approximately 14A1g to l(1
mM) Lis-HC (1(p) (7,5).

Add 2 (11) μl of buffer consisting of 6mM MgCL and 50mM Na(l), plus 50 units of Ps
tl was added and reacted for 4 hours at 37°C to cleave pLIDNA at the Pst1 site. After the reaction product was extracted with phenol-chloroform, ethanol precipitation was performed, and about 13 μg of pLI DNA cut with PStI was recovered. the DNA
Approximately 13 μg was added to TdT buffer at 0.25 mM dG.
50μ of solution containing TP! In addition to T d T (
Add 54 units of P-L Biochemicals) and incubate for 13 minutes at 37°C.
Approximately 14 (dG) chains were added to the 3' end of the I cleavage site.

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

The DNA is 1QmM) Lis-HCj! (pH 7,5>,
6 m M M g Cj! 2 and 60mM
NaCj! In addition to 1 (11) μl of a buffer consisting of pLIDNA, 80 units of HindII was added and incubated at 37° C. for 3 hours, and the pLI DNA was cleaved at the HindII 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., Analytical Biochemistry (Anal, Biochem),
2.295 (1981)) and linker-DNA with oligo (dG) chains (hereinafter simply referred to as linker-D
(abbreviated as NA) was obtained.

Po! prepared above! J (A) About 2 μg of RNA and about 1.4 μg of vector primer at 59 mM) Lis-HCj!
(pH 8,3), 8mM MgCC, 30mM
KCj2.0.3mM DTT, 2mMdNTP
(dATP, dTTP, dC,TP and dcTP) and 10 units of glue bonuclease inhibitor (P-
Solution 2 consisting of L Biochemicals)
It was dissolved in 2.3 μl, 10 units of reverse transcriptase (Seikagaku Corporation) was added, and incubated at 37°C for 40 minutes to synthesize NA complementary to mRNA. The reaction product was extracted with phenol-chloroform. , ethanol precipitation was performed to recover the vector primer DNA to which the RNA-DNA duplex had been added.
Dissolved in μβ, 14 units of T d T (P −L
Biochemicals) and incubated at 37°C for 8 minutes, adding 12 (dC) to the 3' end of the cDNA.
) chains were added.

The reaction product was extracted with phenol-chloroform, and the cDNA-vector primer DNA with the (dC) chain added was recovered by ethanol precipitation. The DNA was diluted with 10mM) Lis-HCj! (pH 7.5), 6mM MgCβ2 and 60mM NaCl, dissolved in 4(11)p(l), added 20 units of HindI, and diluted at 37°C 2
hr and cleaved at the )(ind[
NA-vector primer DNA is the first choice. The DNA0
．． 08 pmole and the linker-DNAQ, l
(3pmole lQmM) Lis-HC1 (pH 7,
5), O, 1M 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
), 4mM MgCL, l OmM(NH<)z
sOs, 0.1 M KCl and 0.1 m Mg-
A reaction solution was prepared with a total volume of 14 (11) μl based on the composition of NAD. 10 units of Escherichia coli DNA ligase (manufactured by New England Biolabs) was added to the reaction solution, and the mixture was incubated overnight at 11°C. 4 each of the reaction solution
Additional components were prepared to give 0 μM dNTP, O, and 15 mM β-NAD, and 5 units of E. coli DNA I
J gauze, 7 units of E. coli DNA polymerase I (P
-L Bio-chemicals) and 2 units of E. coli ribonuclease H (P-L Biochem)
icals) and incubated at 12°C and 25°C for 1 hour each. In the above reaction, circularization of recombinant DNA including cDNA and RN of RNA-DNA duplex
Part A was replaced with DNA, producing a complete double-stranded DNA recombinant plasmid.

Example 3. Selection of recombinant DNA containing eel growth hormone cDNA: Using the recombinant plasmid obtained in Example 2, E. coli C6 (
11) SF8 strain [Cameron: Proceeding of the National Academy of Sciences (Proc, Natl, ^cad, Sc
i, ) USA.

72, 3416 (1975)) using the method of 5cott et al.
Katsuya Shigesada: Cell Engineering, 2.616 (1983))
Transformation was performed according to the following.

Approximately 2.4 (11) colonies obtained were immobilized on nitrocellulose. 2 from the N terminus of eel growth hormone
Synthetic NA corresponding to amino acid sequence 7-32
, that is, 3'5' T A A T A T T T CT T A
A A CT 17mer (T) (G)
(C) (C) (G) (G) (The 3rd base is Δ, T, or G. The 6th base is 8 or G1. The 9th base is T or C1. The 12th base is T or C1. The 15th base is A or G, and when combined, it becomes a mixture of 48 synthetic NAs.) is strongly associated with the P-labeled probe at 38°C.
The selected strain was Grunstein Hognes (Gruns).
Proceedings of the National Academy of Sciences (Prco, Natl, Acad, 5ci)
tlsA, 72°3961 (1975)). The Southern method [Journal of Molecular Biology (Journal of Molecular Biology) was applied to the three obtained bacterial strains.
J, Mat, Rial, ) 98.503° (19
75)), the above probe and a synthetic NA probe corresponding to the amino acid sequence near the C-terminus, namely 5'3' (C) (G) (T) (G) (G) (C) (3rd base is T or C1, the 6th base is A or G1, the 9th base is any of A, T, C, or C, and the 12th base is A or G, resulting in 32 combinations of synthetic NA mixtures. ) A meeting was also confirmed. These plasmids are pEGH
They were named 8, 9, and 15, and because they all had DNA sequences predicted from the amino acid sequence of eel growth hormone, they were considered to contain eel growth hormone cDNA.

Example 4. Base Sequence of Plasmid pEGH15 2 The three plasmids obtained above were digested with various restriction enzymes, and the cleavage map of the cDNA portion was determined. The CDNA restriction enzyme map in plasmid pEGH15 is shown in FIG.

Next, p which showed a strong association with the synthetic NA probe performed in Example 3 and is considered to contain almost full-length cDNA.
The entire nucleotide sequence of the translated region of EGH15 was determined using the Sanger method using M13 phage [Sanger et al.
of the National Academy of Sciences (Proc, Natl, Human Cad.

Sci, ), [ISA, 74.5463 (1977
): 77-Sim (Amersham) M1
3 Cloning and Sea Fencing Handbook
Determined according to handbook). The nucleotide sequences are shown in Table 1. In Table 1, base numbers 1-57 code for the signal peptide, and base numbers 58-627 code for the mature peptide of eel growth hormone.

The amino acid sequence predicted from the cDNAΔ sequence contained in pEGH15 completely matches a part of the amino acid sequence determined from natural eel growth hormone, and the cDNA
was found to encode eel growth hormone (C,H-I) (11g).

1st front gate T: C1 ^ Engineering i I G (ArgPheValGluSerAsnCy=ThrL
E. coli containing eu*4*pEGH15 is ε5cher
ichia coli EEC, HI3 (FERM B
P-824) and was deposited with the Institute of Fine Technology on July 2, 1985.

Example 5. Construction of recombinant plasmid pUPA 1 encoding eel growth hormone (GH-I); Plasmid pEGH153 μg containing DNA encoding eel growth hormone was added to 1QmM) Lis-H (J!
pH 7,5), 7mM MgCj! 2 and 1 (11
) solution containing mM Na (J!) (hereinafter referred to as yio).

Dissolved in 30AI2 (abbreviated as buffer solution), restriction enzyme Ban■
(manufactured by Toyobo Co., Ltd.) and 8 units of restriction enzyme BamH1 (Toyobo Co., Ltd.) were added, and a digestion reaction was performed at 37°C for 2 hours. From the reaction solution, pEGH1 was obtained by LGT method.
The mature eel growth hormone translation region encoded by 5, 3
' - approximately 850 bp including untranslated region and part of the vector
Approximately 0.1 μg of DNA fragment was obtained.

Separately, 2 μg of pGLMl was added to 30 μQ of Y-1 (11).
Dissolve in buffer solution and add restriction enzyme BanI [(manufactured by Toyobo Co., Ltd.)
Add 8 units and restriction enzyme BamHr 8 units, and make 37
Digestion reaction was carried out at ℃ for 2 hours. Approximately 4.0% of the reaction mixture containing PL 0%-tar and cI857 genes was extracted by the LGT method. Approximately 1 inch of OKb DNA fragment was obtained.

On the other hand, in order to add the translation initiation codon ATG necessary for the expression of the DNA encoding mature eel growth hormone, and further to connect the vector DNA and the above DNA, the following DNA I was added.
I synthesized a J-linker.

First, the end-strand DNA 21mer and 15mer were separated using the usual triester method [R, Crea:
Flossing of the National Academy of Sciences (Proc. Natl. Ac.
ad, Sci,).

USA, 75', 5765 (1978)). 21mer and 15mer single-stranded DNA each with 3Qpmole (50mM) Lis-HCl1 (p
H7,5), 10mM MgCj7. , 10mM D
Dissolve in solution 30〃 containing TT and 1mM ATP,
3 units of 4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) were added, and a phosphorylation reaction was carried out at 37°C for 40 minutes.

BanI[-BamH■ fragment of pEGH15 obtained above (approximately 850 bP) 0.03 pmole, pGLM
1 of BamHI-Ban [[ fragment (approximately 4. OKb
) 0. (11) 6pmole to 50mM) Lis-H
Cj! (pH 7,5), 10mM MgCl2.
30ρ solution containing 10mM DTT and 1mM ATP
The above-mentioned synthetic NA phosphorylation reaction solution 1 was added to the solution. Three units of T4DNAIJ gauze (manufactured by Takara Shuzo Co., Ltd.) were added to this mixture, and a binding reaction was performed at 4°C for 18 hours. Using the reaction solution, E. coli strain 294 was transformed, and A
A colony of p11 was obtained, and plasmid DNA was recovered from this colony to obtain pUPA1 shown in FIG. The structure of pUPA 1 is EcoRI, Ban1II.

Hind[II, Ban1l, HpaI, BamHI.

It was digested with Bg l'II, Ps t I, and Xho I, and was recognized by rIi by agarose gel electrophoresis.

Example 6. Production of eel growth hormone (G)l-1) peptide using Escherichia coli containing pUPAl The recombinant plasmid pUPA 1 obtained in Example 5 was used to transform Escherichia coli C6 (11) strain by a conventional method. The obtained AI) colony was added to 10 ml of MCG medium [0
,6%Na211PO4,0,3%KH2PO,,0,
5% NaCj! .

0.1%NH, Il! , 0.5% glucose, 0.5
%casamino acids.

1mM Mg5O<, 4xr/ml vitamin B+
, pH 7.23, and cultured at 30°C for 7 hours.

1. Inoculate 50 ml of MCG medium with the culture solution, and inoculate 3
The cells were cultured at 0°C for 18 hours. The obtained culture solution was diluted with II2M.
Inoculated into CG medium, cultured at 30℃ for 5 hours, then incubated at 42℃ for 2 hours.
The cells were cultured for an hour and then further cultured at 37°C for 41 hours. The divided culture solution was centrifuged at 8 (11) 0 rpm for 10 minutes to collect bacterial cells. Rem this bacterial body! J (Laemml
After suspension in the sample buffer of i), 5DS-polyacrylamide gel electrophoresis was performed, followed by Western blotting [Towbin et al. , 5ci)
, USA.

76, 4350 (1979)), followed by the peroxidase staining method [Fumi Yamabe: Cell Engineering, Yu, 1061 (1983).
): ], the molecular weight is approximately 26. A polypeptide band was detected at the OOO 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 pLIPA1 produced a large amount of eel growth hormone polypeptide.

E. coli containing pUPAl is [! 5cherichia
'col iEUPAI (FERM BP-825)
It was deposited with the Institute of Fine Technology on July 2, 1985.

Example 7゜After culturing eel growth hormone producing bacteria according to Example 6,
8. (11) Collect bacteria by centrifuging at 0 rpm for 10 minutes,
The cells were washed with a buffer (pH 7,5) containing mM NaCl and 33 mM Lys-HCI. The washed bacterial cells were suspended in 5 ml of the above buffer solution and disrupted by ultrasonication at 0°C [Branson Sonic Power Company (Branson
5onic Power Company, Sonifier Cell Disruptor (5onifier)
celldisruptor) 2(11). output control
l) 2. Processed for 10 minutes]. This is 15. (11)
Orpm was centrifuged for 30 minutes to obtain bacterial cell residue. From this bacterial cell residue, the method of Marston et al.
1) (1984)), the eel growth hormone polypeptide was extracted, purified, solubilized, and regenerated, and the growth promoting activity of the eel growth hormone polypeptide was measured by the following method.

Measurement of Fish Growth Hormone Activity Juvenile rainbow trout (average weight 13 g) were divided into groups of 15 fish and reared in a circulating tank at a water temperature of 15°C. The feed was 4% of the body weight per day for the first 9 days, and 3% of the body weight was given in two divided doses for the first 9 days. A small amount of eel growth hormone polypeptide 0.01
After dissolving with N aqueous sodium hydroxide solution, 0.9% aqueous sodium chloride solution was added to make 1×15 layers. This is 1μglt per 1g of body weight! Injected intracavitally. The control group received only 0.9% sodium chloride aqueous solution.

Injections were performed 5 times every 5 days and body weights were measured at the same time.

As a result, it was revealed that the growth hormone of the present invention promotes the growth of rainbow trout.

Example 8. Construction of recombinant plasmid p[JPJ24 encoding eel growth hormone (GH-n) A recombinant plasmid encoding GH-I was constructed as follows.

The recombinant plasmid pUPA12 which encodes eel growth hormone (GH-I) obtained in Example 5 was
Y-1 (11) buffer and restriction enzyme Ban [(
Digestion reaction was carried out using 6 units (manufactured by Toyobo Co., Ltd.) at 37 tons for 2 hours. Add d to the reaction solution at a final concentration of 0.5 mM.
Add ATP and dTTP, then add 4 units of E. coli DNA polymerase I (manufactured by Zenshuzo Co., Ltd.), and incubate at 16°C for 30
The reaction was carried out for minutes. Through this reaction, the ends generated by Ban1l digestion are converted into flat ends in which one base is further removed by the exonuclease activity of DNA polymerase I, exposing the codon encoding the fourth lie residue of GH-I. After phenol and chloroform extraction and ethanol precipitation, the DNA fragments were dissolved in 30 IJjl of Y-1 (11) buffer and treated with 16 units of restriction enzyme PSt for 3
Digestion reaction was carried out at 7°C for 2 hours. From the reaction solution, LGT
By this method, about 0.5 μg of a fragment of about 2.5 Kb containing the mature eel growth hormone translated region, 3'-untranslated region, and E. coli lipoprotein terminator was obtained.

On the other hand, 2 μg of the plasmid vector pPAc1 (see Reference Example 1 for the construction of pPACl), which has a cleavage site for the translation initiation signals ATG and Sph downstream of the PL promoter and the liposome binding site, was added to 30Ii! Y of l
-1 (11) Dissolved in buffer solution, added 6 units of restriction enzyme 5phl (manufactured by Boehringer Mannheim), and heated at 37℃ for 2 hours.
' time digestion reaction was performed.

Add dATP to the reaction solution to a final concentration of 0.5 mM.
.

dCTP, dGTP, and dTTP were added, followed by 4 units of Escherichia coli DNA polymerase ■Klenow fragment (manufactured by Zenshuzo Co., Ltd.), and the reaction was carried out at 16°C for 90 minutes. This reaction allows the 3'
- The overhanging end is DNA polymerase IIK1enOII
Due to the 3'→5' exonuclease activity and 5'-3' repair synthesis activity of the i fragment, it is shaved to a blunt end, and the translation initiation signal ATG is exposed at the end. 65
DNA polymerase I was removed by heat treatment at ℃ for 20 minutes.
- After inactivating the Klenow fragment, the restriction enzyme Pst
16 units were added and the digestion reaction was carried out at 37°C for 2 hours. From the reaction solution, cI857 gene, PL
promoter.

Approximately 0.2 Kb fragment containing translation initiation signal ATC
．． 5■ is the 1st prize.

Each of these DNA fragments thus obtained was 0.2
d of 50mM Tris-HCj! (pH 7.5),
10mM MgCL, 10mM DTT and 1m
M Dissolved in 30III of a solution containing ATP, T4
DNA! Add 30 units of J gauze (manufactured by Zenshuzo Co., Ltd.),
The binding reaction was carried out at 4°C for 16 hours. E. coli strain 294 was transformed using the reaction mixture to obtain A p l colonies, plasmid DNA was collected from these colonies, and the fourth
pUPJ24 shown in the figure was obtained. The structure of pLIPJ24 is the restriction enzyme Pstl.

It was cleaved with BanII[, N5iI, Bgll[, and BamHI, and confirmed by agarose gel electrophoresis.

In addition, pUPJ 24 is GH after the translation start signal ATG.
-1 after the fourth amino acid residue, i.e. GH-II
The Sanger method using M13 phage, [Sanger
) et al.: Proceedings of the National Academy of Sciences (proc.

Natl, ^cad, Sci,), USA, 74
．． 5463 (1977); Amer Jam (Amer
sham) M13 Cloning and/- Fencing Handbook (C]oniBand sequ
Encing handbook)].

Example 9. Production of eel growth hormone (C, H-11) peptide using Escherichia coli containing pUPJ24 Using the recombinant plasmid pUPJ 24 obtained in Example 8, Escherichia coli strain 294 was transformed by a conventional method. One colony of the obtained A+) was inoculated into 5 ml of MCG medium and cultured at 30°C for 16 hours. 1 of the obtained culture solution
(11) I was inoculated into 5 ml of MCG medium and incubated at 30°C for 2 hours.
After culturing for an hour, the cells were cultured at 42°C for 2 hours. The resulting culture solution was centrifuged at 8(11)0 rpm for 10 minutes to collect the bacterial cells. After suspending this bacterial cell in Laemmli sample buffer, 5DS-polyacrylamide gel electrophoresis was performed and stained with Coumasieve IJ IJ Ant Blue, and a polypeptide band was found at the site with a molecular weight of approximately 25 and (11)0. was detected.

This band was not present when E. coli not containing the plasmid was used. Similarly, after suspending the collected bacterial cells in a sample buffer of REM U (Lae+nm1i), 5DS-polyacrylamide gel electrophoresis was performed, and Western blotting [Tobin
) et al; Proceedings of the National Academy of Sciences (Proc. Natl.
Acad, Sci,), USA 76,
4350 (1979) After E, Belokindase staining method [Tabe Fumi: Cell Engineering, 2, 1061. (1
983B, also has a molecular weight of about 25. (11) A polypeptide band was detected at the 0 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 pUPJ 24 produced a large amount of eel growth hormone polypeptide.

Reference example 1. P, plasmid vector p with translation initiation signal ATC and Sphr cleavage site downstream of the promoter and liposome binding site.
Construction of PAc1 To construct a recombinant plasmid encoding eel growth hormone (GH-II) described in the present invention, pPAc
I created l.

Plasmid vector pTrS3 (Japanese Unexamined Patent Publication No. 59-6729
7) Dissolve 2■ in 30AI Y-1 (11) buffer,
4 units of restriction enzyme Band (manufactured by Toyobo Co., Ltd.) and 14 units of restriction enzyme Pst were added, and a digestion reaction was performed at 37°C for 2 hours. Translation initiation signal AT is extracted from the reaction solution by LGT method.
Approximately 2.9 K b of D containing G and Sph■ cleavage sites
Approximately 1 μg of NA fragment was obtained.

On the other hand, pGLMl 2 rivers are 304 y-io.

(Dissolve in 1 solution, add 4 units of restriction enzyme 13anIIr (manufactured by Toyobo Co., Ltd.) and 4 units of restriction enzyme PStI,
Digestion reaction was carried out at 37°C for 2 hours. LGT from the reaction solution
Approximately 4.0% containing the PL promoter and cr857 gene by the method. Approximately 1 g of Q K b DNA fragment was obtained.

0.1g of each of these DNA fragments at 50mM)
Squirrel-HCj! (pH 7,5), 10mM MgCC
, 10mM DTTi, and 1mM ATP were dissolved in 30μ of a solution containing T4DNA IJ gauze (Takara Shuzo Co., Ltd. rM > 30 units), and a binding reaction was performed at 4°C for 16 hours. The strain was transformed to obtain a colony of Δp1, and plasmid DNA was recovered from this colony to obtain pPACl shown in Figure 5.The structure of pPACl is Ps t I, Sph i Ba.
nII [, Sa jl! It was cut with I and confirmed by agarose gel electrophoresis.

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

[Brief explanation of drawings]

Figures 1 (1) and (2) outline the process of cDNA synthesis by the Okayama-Barb method and the construction of a recombinant plasmid containing the DNA. FIG. 2 shows a restriction enzyme map of the cDNA encoding eel growth hormone contained in pEGH15. FIG. 3 shows the construction process of recombinant plasmid pUPΔl. FIG. 4 shows the construction process of recombinant plasmid pUPJ24. FIG. 5 shows the construction process of recombinant plasmid pPAc1. 1st (1) HindlII ¥I L+ko (2) ccc ym+++++ ---
-aceAAA (:1” ” ” ” cCCC
AAAA--------- DNA! J-Bi Figure 5 Procedural Amendment (Voluntary) Procedural Amendment (Method ヱ℃) July 1985 S Usu Toku T1 Director General 1. Indication of Tenancy 1988 Patent Application No. 74061 2. Invention Name of the fish growth hormone gene and the polypeptide 3 encoded by the gene, and its relationship to the case of the person making the amendment Patent applicant Postal code: 1 (11) Address: 6-1 Otemachi-chome, Chiyoda-ku, Tokyo Name: (102) Kyowa Hakko Kogyo Co., Ltd. (TE L:
03-201-7211 extension 3391λ. June 4, 1986 (shipped on June 24, 1986) 5.
Target of amendment July 2, 1986

Claims (11)

[Claims] (1) A fish growth hormone polypeptide having the peptide sequence shown in Table 1. (2) The polypeptide according to claim 1, wherein the fish growth hormone is a fish growth hormone belonging to the order Apodidae. (3) DNA encoding a growth hormone polypeptide of a fish belonging to the order Apodidae. (4) The DNA of claim 3, in which the fish growth hormone polypeptide has the peptide sequence shown in Table 1.
. (5) Recombinant DNA incorporating DNA encoding a growth hormone polypeptide of a fish belonging to the order Apodidae
. (6) The recombinant DNA of claim 5, wherein the fish growth hormone polypeptide has the peptide sequence shown in Table 1. (7) The recombinant DNA of claim 5, named plasmid pEGH15. (8) Recombinant DNA incorporating DNA encoding a growth hormone polypeptide of a fish belonging to the order Apodidae
Microorganisms including. (9) The microorganism according to claim 8, wherein the fish growth hormone polypeptide has the peptide sequence shown in Table 1. (10) The microorganism according to claim 8, wherein the microorganism belongs to Escherichia coli. (11) Recombinant DNA incorporating DNA encoding a growth hormone polypeptide of a fish belonging to the order Apoda, which is a ray-finned fish.
A method for producing a fish growth hormone polypeptide, which comprises culturing a microorganism containing A in a nutrient medium, accumulating a fish growth hormone polypeptide in the culture, and collecting the polypeptide from the culture. .