JPH05268969A - New growth hormone polypeptide of fishes - Google Patents

Abstract

PURPOSE:To obtain a new DNA useful for producing a growth hormone of fishes. CONSTITUTION:The objective DNA is capable of coding a growth hormone of fishes having a peptide sequence of the formula. The whole RNA is prepared from a salmon hypophysis, passed through an oligo(dT)cellulose column to separate a poly(A)RNA, which is then used as a template to synthesize a double- stranded DNA with a reverse transcriptase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding a novel growth hormone polypeptide of fish, a recombinant DNA incorporating the DNA, and a microorganism containing the recombinant DNA. The fish growth hormone is expected to have a wide range of applications in the field of fish farming industry.

[0002]

Mammalian growth hormone is produced in the pituitary gland, but its activity and structure are known. For example, for human growth hormone, the Journal of American Chemical Society (J.Am.Che) was published by UJ Lewis et al.
m.Soc.), 80 , 4429 (1958), by AS Hartree in the Biochemical Journal (Biochem.J.), 100 , 754 (1966), C.H.
Archi Biochem. Biophys. (Suppl.)], 1 , 327 (1) by Lee (CHLi) et al.
962).

There are many reports that the growth hormones of fish have been isolated so far, but few of them are reliable in their physiological activity and protein chemical properties. Examples of reliable reports include: Isolation example from Tilapia: S. W. Farmer et al., General and Comparative Endocrinology (Gen. Comp. Endocrin.,
30 , 91 (1976). Isolation example from sturgeon: S. W. Farmer et al., Endocrinolii.
ogy), 108 , 377 (1981). Isolation example from carp: AF Cook
Gen. Comp. Endocrin., 50 , 335 (1983). On the other hand, for the growth hormone gene of mammals, the rat growth hormone gene [P.S.
eeburg) et al: Nature 270 , 486 (1977)
], Bovine and porcine growth hormone genes [PH Seeburg et al .: D.N.
Ray (DNA), 2 , 37 (1983)], human growth hormone gene [JA Martial et al .: Science, 205 , 602 (1979)], etc. The growth hormone gene has also been isolated by the present inventors [Japanese Patent Application Nos. 59-134536 and 59-213360]. The fish growth hormone polypeptide encoded by the gene was previously prepared from salmon pituitary gland by Kawauchi et al. And was confirmed to have a growth promoting effect in teleost fish. -68670], the N-terminal (40) and C-terminal (21) amino acid sequences were completely identical.

[0004]

Since the fish growth hormone has a fish growth promoting effect, it is useful as a composition for a fish feed, but the amount of the fish supplied from the pituitary gland is limited. There is. Therefore, it is desired to develop a method for inexpensively supplying a large amount of fish growth hormone.

[0005]

The present inventors have found that recombinant D
A study was conducted on a method for producing fish growth hormone by the NA technique. As a result, we succeeded in collecting a DNA complementary to a fish growth hormone polypeptide, which can be used for producing a fish growth hormone, and producing a recombinant DNA and a microorganism containing the DNA. That is, messenger RNA (mRNA) was extracted from salmon pituitary gland, DNA (cDNA) complementary thereto was synthesized, and then a DNA probe corresponding to the amino acid sequence near the N-terminal of salmon growth hormone was synthesized. CDNA that hybridizes with DNA
By selecting A, we succeeded in cloning the salmon growth hormone gene, and already applied for a patent (Japanese Patent Application Nos. 59-134536 and 59-213360). Furthermore, in the process of examining several salmon growth hormone genes in detail, it was found that there was another gene highly correlated with the one previously filed for patent, and the whole nucleotide sequence of its cDNA was determined. The present inventors further proceeded with research, and by culturing a microorganism containing a recombinant DNA in which a DNA encoding the growth hormone of the novel salmon was cultured, a salmon growth hormone polypeptide was produced and accumulated in a large amount in the culture. The present invention has been completed and the present invention has been completed.

The present invention will be described in detail below. The present invention provides novel fish growth hormone polypeptides, particularly polypeptides having the peptide sequence set forth in SEQ ID NO: 1. The polypeptide can be produced using recombinant DNA techniques as described below. That is, using the mRNA of fish growth hormone as a template, a 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 novel growth hormone genes, especially in bacteria such as Escherichia coli. The microorganism having the recombinant plasmid is useful for inexpensively producing a large amount of novel fish growth hormone polypeptide in large quantities.

Accordingly, the present invention relates to a DNA encoding a novel growth hormone polypeptide of fish, a recombinant DNA incorporating the DNA, a microorganism containing the recombinant DNA, and a novel growth hormone polypeptide using the microorganism. Provide a manufacturing method. The DNA of the present invention and the recombinant plasmid are prepared by the following general procedure. Total RNA was prepared from chum salmon pituitary gland and oligo (dT) cellulose [ol
RNA [poly (A) RNA] having polyadenylic acid [poly (A)] is separated by passing through an igo (dT) cellulose column.

Double-stranded DNA is synthesized by reverse transcriptase using this poly (A) RNA as a template. Recombinants can be obtained by inserting the synthetic DNA into vector DNA such as E. coli plasmid DNA using in vitro DNA recombination technique. Next, the method for producing the DNA and recombinant plasmid of the present invention will be specifically described.

The pituitary gland is removed from the captured chum salmon and immediately frozen in liquid nitrogen. This frozen pituitary gland contains guanidium isothiocyanate.
Hiocyanate) is added and crushed to solubilize. Then CsC
l Cytoplasmic RN as a precipitate
Get A. Alternatively, LiCl can be added to the solubilized product of guanidinium isothiocyanate to precipitate and recover only RNA.

The extracted RNA was dissolved in a high salt (for example, 0.5 M) solution of NaCl or KCl, and the oligo (d
T) It is passed through a cellulose column to adsorb mRNA having poly (A) onto the column. Water, 10 mM Tris-
Elute with a low salt solution, such as HCl buffer, to isolate mRNA with poly (A). Hereafter, the method of Okayama-Berg [Okayama and
Berg (Okayama &Berg); (. Mol.Cell.Biol) Molecular and Cellular Baiorojii 2, 161 (198
2)], cDNA synthesis and its integration into a vector are performed.

First, a vector primer is synthesized. As a vector, for example, pCDV1 may be used in an appropriate solution such as Tris-HCl buffer (eg pH 7.5, 10 mM), M
gCl ₂ (eg 6 mM), NaCl (eg 10 m
M) is treated with KpnI to cleave the KpnI site of pCDV1. This DNA is Tris-HCl
Buffer (eg pH 6.8, 30 mM), sodium cacodylate (eg 140 mM), CoCl ₂ (eg 1 m
M), dithiothreitol (eg 0.1 mM) and dTTP (eg 0.25 mM) incubate with terminal deoxynucleotidyl transferase at constant temperature (eg 37 ° C.) for constant time (eg 20 minutes) to obtain both vector DNA. About 60 thymidyl residues are added to the 3'end. Further, this DNA is added to Tris-HCl buffer (eg pH 7.5, 10 mM), M
gCl ₂ (eg 6 mM), NaCl (eg 10 mM)
After cutting with EcoRI in a solution containing 0 mM, low melting point agarose gel electrophoresis [Lars Wisslander (Lars W
ieslander): Fractionation by Analytical Biochemistry, 98 , 305 (1979)] to recover a fragment of about 3.1 kilobases. The DNA is then added to a high salt concentration of NaCl or KCl (eg.
5M) solution and passed through a poly (dA) cellulose column to adsorb only the vector primer molecule having poly (T) to the column. Water, 10 mM Tris-HCl
Elute with a low salt solution such as a buffer to isolate only poly (T) -added vector primer molecules.

Next, linker DNA is synthesized. For example, pL1 DNA is treated with PstI in an appropriate solution such as Tris-HCl buffer (eg pH 7.5, 10 mM), MgCl ₂ (eg 6 mM), NaCl (eg 50 mM) to cleave the PstI site of pL1. To do. This DNA is treated in the same manner as in the case of vector primer synthesis except that dGTP is added instead of dTTP, and about 15 oligo (dG) chains are added. The DNA in a suitable solution, such as Tris-HCl buffer (eg pH 7.5, 10 mM), MgCl ₂ (eg 6 mM),
HindII in a solution containing NaCl (eg 60 mM)
Cut at I. A DNA fragment of about 0.5 kilobases is fractionated by agarose gel electrophoresis and recovered with DEAE paper. Thus, the linker DNA is obtained.

CDNA synthesis is carried out using the poly (A) RNA, vector primer and linker DNA obtained as described above. Poly (A) RNA, vector primer DNA
Tris-HCl buffer (eg, pH 8.3, 50 m
M), MgCl ₂ (eg 8 mM), KCl (eg 30 mM), dithiothreitol (eg 0.3 m)
M), dATP, dTTP, dCTP, dGTP (for example, 2 mM each), the reverse transcriptase is reacted at a constant temperature (for example, 37 ° C.) for a constant time (for example, 40 minutes). At the 3'end of the thus obtained RNA-DNA duplex, oligo (d) was added by the same operation as the condition of adding (dT) strand to the vector primer except that dTTP was changed to dCTP.
C) Add about 15 chains.

This DNA was added to Tris-HCl buffer (eg pH 7.5, 10 mM), MgCl ₂ (eg 6 mM),
Hind in a solution containing NaCl (eg 60 mM)
Cut at III. This DNA is mixed with the previously prepared linker DNA, and the mixture is mixed with Tris-HCl buffer (for example, pH 7.
5,20 mM), MgCl ₂ (eg 4 mM), (N
H ₄ ) ₂ SO ₄ (eg 10 mM), KCl (eg
0.1 M), β-nicotinamide adenine dinucleotide (β-NAD) (eg 0.1 mM) in a solution containing E. coli.
For a certain time (eg 16 hours) with DNA ligase,
Incubate at constant temperature (eg 12 ° C). In this way, circularization of the cDNA and the linker DNA is performed. DATP, dTTP, dGTP, dC in this reaction solution
TP was added to each so that the final concentration was 40 μM, and E. coli D was added.
By adding NA ligase, Escherichia coli DNA polymerase I, and Escherichia coli ribonuclease H to convert the RNA portion into DNA, a recombinant plasmid containing the complete double-stranded cDNA is obtained.

Using the recombinant plasmid thus obtained, Escherichia coli, for example, Escherichia coli c600SF8 strain, can be obtained, for example, by the method of Scott et al. [Katsuya Shigeta: Cell Engineering.
2 , 616 (1983)]. Since the ampicillin resistance gene is present on the recombinant plasmid obtained above, the transformed Escherichia coli shows ampicillin resistance. The following approach is commonly used to select the strains carrying the novel recombinant plasmid DNA having a gene showing complementarity to growth hormone mRNA in fish from these ampicillin resistant (Ap ^r) strain. That is, the transformant obtained above is immobilized on a nitrocellulose filter and is allowed to associate with a synthetic DNA probe having a DNA sequence predicted from the amino acid sequence of a known chum salmon growth hormone, and a substance that strongly associates is selected [ Grunstein-Hogness Method, Proceeding of the National Academy of
Science (Proc.Natl.Acad.Sci)., USA., 72 , 3961 (1
975)]. The probe DNA is synthesized by a usual triester method [Journal of American Chemical Society (J. Am. Chem. Soc.), 97 , 7327 (1975)]. Selection with synthetic DNA probes
rn) et al. [J. Mol. Biol.), 98 , 503 (1975)], and the method of chum salmon growth hormone m
Recombinant plasmid DNA having a gene that is complementary to RNA can be identified.

An example of the recombinant plasmid thus obtained is pSGH14. 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: The DNA was excised from a plasmid containing the DNA encoding salmon growth hormone, and this was cut into vector D
By incorporating into NA, introducing the obtained recombinant DNA into a microorganism, and culturing the obtained transformant, the salmon growth hormone polypeptide is produced and accumulated in the culture,
A salmon growth hormone polypeptide can be produced by collecting this.

A preferable example of the plasmid containing the DNA encoding salmon growth hormone is pSGH14. As the vector DNA, the inserted D
Any NA can be used as long as it can be expressed in a microorganism. Preferably, it has a suitable promoter, for example, a promoter of tryptophan (trp) system, lactose (lac) system, PL system, and the like, into which DNA can be inserted downstream, and an internal Shine-Dalgarno sequence (hereinafter abbreviated as SD sequence). And a translation initiation codon (ATG) are used at an appropriate distance, for example, 6 to 18 base pairs. As a specifically suitable vector DNA, the plasmid pGEL1 can be mentioned. pGEL1 is the plasmid shown in FIG. 4, and E. coli containing it is Escherichia coli IGE.
1984 as L1 (FERM BP-629)
Microbiology Industrial Technology Research Institute (AIST), 6th of March
Has been deposited with. Recombination between the DNA encoding the polypeptide and the vector DNA is carried out by using a restriction enzyme.
After digestion of A, it can be carried out using a general recombinant DNA technique in which T4 DNA ligase is used for ligation.

In the case of pSGH14 and pGEL1 shown as specific examples, two-step construction is performed as shown in FIG. Ie pS
From GH14, an MboII-PvuII fragment encoding the vicinity of the N-terminal of the salmon growth hormone mature peptide and a PvuII-HindIII fragment containing the remaining part of the cDNA and the vector part are separately obtained. Meanwhile, the following synthetic DNA linker is prepared.

[0019]

[Chemical 1]

The above DNA fragment and the synthetic DNA linker are ligated with T4 DNA ligase to obtain the recombinant plasmid pSGHIIB9 shown in FIG. Next, a HindIII-BamHI digested fragment encoding a salmon growth hormone mature peptide was obtained from pSGHIIB9, and a HindIII-BamHI digested fragment containing a tryptophan promoter was obtained from pGEL1. The above two DNs
The A fragment is ligated with T4 DNA ligase to obtain the recombinant plasmid pSGHIIC2 shown in FIG. This plasmid has a form in which a region encoding mature salmon growth hormone is linked downstream of the tryptophan promoter.

The reaction conditions in the above recombinant technique are generally as follows. For the digestion reaction of DNA with restriction enzymes, 0.1 to 20 μg of DNA is usually used for 2 to 200 m
M (preferably 10-40 mM) Tris-HCl (p
H 6.0 to 9.5, preferably pH 7.0 to 8.0), 0 to 200
mM NaCl, 2-30 mM (preferably 5-10 m
M) in a reaction solution containing MgCl ₂ and a restriction enzyme of 0.1 to 1
Using 00 units (preferably 1 to 3 units for 1 μg of DNA), the reaction is carried out at 20 to 70 ° C. (the optimum temperature depends 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.

The DNA fragment generated by restriction enzyme digestion was purified by low melting point agarose gel electrophoresis [L. Wieslander: Analytical Biochemistry 98 , 305 (197).
9), hereinafter referred to as LGT method] or polyacrylamide gel electrophoresis. The binding reaction of DNA fragments is
2 to 200 mM (preferably 10 to 40 mM) Tris-HCl (pH 6.1 to 9.5, preferably pH 7.0 to 8.
0), 2 to 20 mM (preferably 5 to 10 mM) Mg
Cl ₂ , 0.1 to 10 mM (preferably 0.5 to 2.0 mM)
Of ATP and 1 to 50 mM (preferably 5 to 10 mM) of dithiothreitol in a reaction solution containing 0.3 to 10 units of T4 DNA ligase at 1 to 37 ° C. (preferably 3).
15 minutes to 72 hours (preferably 2 to 20)
Time) The recombinant plasmid DNA generated by the ligation reaction may be transformed, if necessary, by the transformation method of Cohen et al.
SN Cohen et al: Proceeding ·
Of the National Academia of Science (Proc. Natl. Acad. Sci.), USA 69 , 2110 (1972)].

Isolation of the recombinant plasmid DNA from E. coli is carried out by the method shown in Example 1 or by Birnboim et al. [HC Birnboim et al .: Nucleic Acid].・ Research (Nucleic Acids Res.) 7 , 1513
(1979)] and the like. 1 to 1 for plasmid DNA
After digestion with 0 kinds of restriction enzymes, the cleavage site is examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis. Further, when it is necessary to determine the nucleotide sequence of DNA, the Maxam-Gilbird method [Procedure of the National Academia of Science (Pr
oc. Natl. Acad. Sci.), 74 , 560 (1977)] or M13
Sanger method using phage [Sanger (Sa
nger) et al. Proceeding of the National Academy of Science (Proc. Natl. Acad. Sc
i.) USA, 74 , 5463 (1977); Amersham
M13 Cloning and Sequencing Handbook]. Recombinant plasmid DN under the above conditions
A can be produced.

The fish growth hormone peptide of the present invention can be produced as follows. That is, Escherichia coli K-12 HB101 was transformed with a plasmid (for example, pSGHIIC2), and pSGHII was selected from ampicillin-resistant colonies.
Escherichia coli having C2 is selected. By culturing Escherichia coli having pSGHIIC2 in a medium, a fish growth hormone polypeptide can be produced in the culture.

The medium used here may be either a synthetic medium or a natural medium as long as it is suitable for the growth of Escherichia coli and the production of the growth hormone polypeptide of fish.
As the carbon source, glucose, fructose, lactose, glycerol, mannitol, sorbitol, etc., and as the nitrogen source, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ ,
Casamino acids, yeast extract, polypeptone, meat extract, bactotryptone, corn steep liquor, etc. are other nutrient sources such as K ₂ HPO ₄ , KH ₂ PO ₄ ,
NaCl, MgSO ₄ , vitamin B ₁ , MgCl _{2 and the} like can be used.

The culture has a pH of 5.5 to 8.5, a temperature of 18 to 40 ° C.
It is carried out by aeration and stirring culture. Since chum salmon growth hormone polypeptide accumulates in the cultured cells in 5 to 90 hours of culturing, the cells are collected from the culture, the cells are treated with lysozyme, and then frozen and thawed repeatedly to disrupt the cells. The polypeptide is collected from the supernatant obtained by centrifugation according to the usual method for extracting a polypeptide.

For the detection of the polypeptide, the cultured bacterial cells are directly analyzed by a Laemmli sample buffer [Lemuri (L
aemmli), Nature, 227 , 680 (1970))
After heating and thawing, the mixture is applied to SDS-polyacrylamide gel [Laemmli method: ibid.] And stained with Coomassie Brilliant Blue. Examples of the present invention will be shown below.

[0028]

【Example】

Example 1 Preparation of poly (A) RNA from chum salmon pituitary: guanidinium thiocyanate-cesium chloride method from chum salmon pituitary [Maniatis
) Et al., Molecular Cloning (Molecular Clo
ning), p196, Cold Spring Harbor (Cold
Spring Harbor); Katsuya Shigetada, Cell Engineering, 2 , 616 (19)
83)], RNA having poly (A) was prepared as follows.

2 g (about 30 individuals) of frozen pituitary gland of chum salmon was added to 4 M guanidinium thiocyanate, 0.5% sarcosine, 5 mM sodium citrate (pH 7) and 0.1.
It was solubilized by crushing with a Teflon homogenizer (5 rpm) in 10 ml of a solution containing Mβ-mercaptoethanol. Pass this homogenate through an 18G needle several times to DN
A was divided. 5.7M CsCl, 0.1M EDTA
1.2 ml of each (pH 8) solution was dispensed into an ultracentrifuge tube, and the homogenate was overlaid. Hitachi RPS40
After centrifugation at 35,000 rpm for 15 hours in a rotor, RNA was recovered as a precipitate. The RNA precipitate was dissolved in 10 ml of a Tris-HCl (pH 8.0) solution containing 1 mM EDTA, extracted with phenol-chloroform, and then recovered by ethanol precipitation. About 1 mg of the obtained RNA was added to 10 mM Tris-
It was dissolved in 1 ml of a solution consisting of HCl (pH 8.0) and 1 mM EDTA. Incubate at 65 ° C for 5 minutes,
0.1 ml of 5M NaCl was added. Mix the mixture with oligo (dT)
Cellulose column (PL Biochemicals) chromatography (column volume 0.5 ml) was applied. The mRNA having the adsorbed poly (A) was treated with 10 mM Tris-HCl.
Elute with a solution consisting of (pH 7.5) and 1 mM EDTA.
Fractionation was performed in 2 ml portions. The 3rd to 5th fractions were collected to obtain about 10 μg of mRNA having poly (A).

Example 2 cDNA synthesis and insertion of the DNA into a vector: Okayama-Berg method [Molecular and Cellular Biol., 2 , 161 (1982). ], The cDNA
Was synthesized and a recombinant plasmid incorporating it was constructed. The outline of the process is shown in FIGS.

PCDV1 [Okayama & Berg: Molecular and Cellular Biol., 3 , 280 (198)
3)] 400 μg of 10 mM Tris-HCl (pH 7.
5), added to 300 μl of a solution consisting of 6 mM MgCl ₂ and 10 mM NaCl, and 500 units of Kpn
I (manufactured by Takara Shuzo Co., Ltd., unless otherwise specified, all restriction enzymes were manufactured by Takara Shuzo Co., Ltd.) was added, reacted at 37 ° C. for 6 hours, and cleaved at the KpnI site in the plasmid. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation. About 200 μg of the KpnI-cleaved DNA was added to 40 mM
Sodium cacodylate, 30 mM Tris-HCl (pH
6.8), 1 mM CaCl ₂ and 0.1 mM dithiothreitol (hereinafter abbreviated as DTT) to a buffer solution (hereinafter abbreviated as TdT buffer solution) to which dTTP was added at 0.25 mM, and added to 200 μl of the solution. Furthermore, 81 units of terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT) (manufactured by PL Biochemicals)
Was added and reacted at 37 ° C. for 11 minutes. Where pCD
Approximately 67 poly (dT) chains were present at the 3'end of the KpnI cleavage site of V1.
Individually added. The solution was subjected to phenol-chloroform extraction and ethanol precipitation to obtain poly (dT) chain-added pC.
About 100 μg of DV1 DNA was recovered. 10 times the DNA
mM Tris-HCl (pH 7.5), 6 mM MgCl ₂ ,
To 150 μl of a buffer consisting of 100 mM NaCl was added 360 units of EcoRI, and the mixture was reacted at 37 ° C. for 2 hours. About 3.1K after treating the reaction product by LGT method
The DNA fragment of b was recovered and added with about 60 μg of poly (dT) chain.
CDV1 was obtained. The DNA was added to 10 mM Tris-HCl.
Solution consisting of (pH 8.0) and 1 mM EDTA 50
It was dissolved in 0 μl, incubated at 65 ° C. for 5 minutes, ice-cooled, and 50 μl of 5M NaCl was added. The mixture is oligo (dA) cellulose column manufactured by Collaborative Research)
Chromatography. Those with sufficient poly (dT) chain length are adsorbed on the column, which is then added to 10 mM Tris-HCl (p
Elution was performed with a solution consisting of H8.0) and 1 mM EDTA to obtain 27 μg of pCDV1 (hereinafter abbreviated as vector primer) to which poly (dT) chain was added.

Next, the linker DNA is prepared. pL1 [Okayama & Ber
g): Molecular and cellular biology (Mol. Cell.Biol.), 3 , 280 (1983)] Approximately 14 μg of 10
To 200 μl of a buffer consisting of mM Tris-HCl (pH 7.5), 6 mM MgCl ₂ and 50 mM NaCl, 50 units of PstI was further added, reacted at 37 ° C. for 4 hours, and cleaved at the PstI site in pL1 DNA.
The reaction product was extracted with phenol-chloroform, precipitated with ethanol, and the pL1 DNA cleaved with PstI was approximately 13 μm.
g was recovered. About 13 μg of the DNA was added to 50 μl of a solution containing dGTP at a final concentration of 0.25 mM in TdT buffer, and 54 units of TdT (PL Biochemicals) was further added.
After incubation at 37 ° C for 13 minutes, about 14 (dG) chains were added to the 3'end of the PstI cleavage site of pL1. After extraction with phenol-chloroform, DNA was recovered by ethanol precipitation. 100 μl of 10 mM Tris-HCl
(PH 7.5), 6 mM MgCl ₂ and 60 mM N
To 100 μl of a buffer containing aCl, 80 units of HindIII was further added, and the mixture was incubated at 37 ° C. for 3 hours,
It was cut at the HindIII site of pL1 DNA. The reaction product was fractionated by agarose gel electrophoresis to give a D of about 0.5 Kb.
The NAAE fragment is processed by the DEAE paper method [Dretzen]
Et al., Analytical Biochemistry (Anal. Bioc
hem.), 112 , 295 (1981)], and a linker DNA with an oligo (dG) chain (hereinafter simply referred to as linker DNA) was obtained.

About 2 μ of the poly (A) RNA prepared above
g, about 1.4 μg of vector primer was added to 50 mM Tris-
HCl (pH 8.3), 8 mM MgCl ₂ , 30 mM
KCl, 0.3 mM DTT, 2 mM dNTP (dAT
P, dTTP, dGTP and dCTP) and 10 units of ribonuclease inhibitor (PL Biochemi
Cals) solution (22.3 μl) was added, 10 units of reverse transcriptase (Seikagaku Corporation) was added, and the mixture was incubated at 37 ° C. for 40 minutes to synthesize a DNA complementary to mRNA. The reaction product was subjected to phenol-chloroform extraction and ethanol precipitation to recover a vector primer DNA having an RNA-DNA double strand added. 6 the DNA
TdT containing 6 μM dCTP and 0.2 μg poly (A)
Dissolve in 20 μl of buffer and add 14 units of TdT (P-L
Biochemicals) was added and incubated at 37 ° C. for 8 minutes to add 12 (dC) chains to the 3 ′ end of cDNA.
The reaction product was extracted with phenol-chloroform, and the (dC) chain-added cDNA-vector primer DNA was recovered by ethanol precipitation. The DNA was added to 10 mM Tris-
HCl (pH 7.5), 6 mM MgCl ₂ and 60 m
It was dissolved in 400 μl of a solution containing M NaCl, 20 units of HindIII was added, and the mixture was incubated at 37 ° C. for 2 hours and cleaved at the HindIII site. The reaction product was extracted with phenol-chloroform and precipitated with ethanol to give 0.5 pmol.
(DC) chain-added cDNA-vector primer DNA was obtained. 0.08 pmole of said DNA and said linker DNA
0.16 pmol was added to 10 mM Tris-HCl (pH 7.5),
Dissolve in 40 μl of a solution consisting of 0.1 M NaCl and 1 mM EDTA, and add 1 each at 65 ° C., 42 ° C., 0 ° C.
Incubated for 0 minutes, 25 minutes, and 30 minutes. 20 mM
Tris-HCl (pH 7.5), 4 mM MgCl ₂ , 10 mM (N
H ₄ ) ₂ SO ₄ , 0.1 M KCl and 0.1 mM β-NAD
The reaction solution was prepared so that the total volume would be 400 μl. 10 units of E. coli DNA ligase (New
England Biolabs) was added and incubated overnight at 11 ° C. The reaction solution was mixed with 40 μM of dNTP, 0.15
The components were additionally prepared to be mM β-NAD, 5 units of E. coli DNA ligase, 7 units of E. coli DNA polymerase I (PL Biochemicals) and 2 units of E. coli ribonuclease H (PL Biochemicals) were added, and 12 Incubation was carried out at 1 ° C and 25 ° C for 1 hour respectively. In the above reaction, circularization of the recombinant DNA containing cDNA and replacement of the RNA portion of the RNA-DNA double strand with DNA produced a recombinant plasmid of complete double-stranded DNA.

Example 3 Chum Salmon Growth Hormone cDNA
Selection of Recombinant DNA Containing: Using the recombinant plasmid obtained in Example 2, E. coli c600SF8 strain [Cameron (Camero
n): Proceeding of the National Academy of Science (Proc. Natl. Acad. Sci.) US
A, 72 , 3416 (1975)] was transformed according to the method of Scott et al. [Katsuya Shigetada: Cell Engineering , 2,616 (1983)]. Of the approximately 10,000 colonies obtained, 4800 were fixed on nitrocellulose. N of chum salmon growth hormone
Synthetic DNA corresponding to the 23rd to 28th amino acid sequence from the end, that is,

[0035]

[Chemical 2]

(3rd base is A or G, 9th is T
Or C, the 12th is C or T, and the 15th is C or T, and they are combined to form a mixture of 16 synthetic DNAs), and 8 strains strongly associated with the probe labeled with ³² P at 40 ° C. were selected. [Grunstein-Hogness (Grun
stein-Hogness method, Proceeding of the National Academy of Science (Proc.
Natl. Acad. Sci.) USA, 72 , 3961 (1975)]. About the obtained 8 strains, the method of Southern [J. Mol. Bio
l.), 98 , 503 (1975)], and a synthetic DNA probe corresponding to the above-mentioned probe and the amino acid sequence near the C-terminal.

[0037]

[Chemical 3]

(The third base is C or T, the sixth base is A
Or G, the 9th is any one of A, T, G, and C, the 12th is G or A, and 32 combinations D are combined.
A mixture was also confirmed. These plasmids were named pSGH1,3,6,8,9,10,14,17, and all contain the growth hormone cDNA because they have the DNA sequence predicted from the amino acid sequence of chum salmon growth hormone. Was thought to be.

Example 4 Nucleotide sequence of the plasmid pSGH14: The 8 types of plasmids obtained above were digested with various restriction enzymes to determine the cleavage map of the cDNA portion. The obtained plasmids can be classified into 3 groups based on the positions of the restriction enzyme sites, and the pSGH 1, 6, 9, 10, 17 group, the pSGH 3 group, and the pSGH 3 group.
Separated from GH8,14 group. The restriction enzyme map of each group is shown in FIG.

Of these, plasmids of the pSGH1 group,
Especially for pSGH1, the entire nucleotide sequence has already been determined, and the amino acid sequence predicted from the cDNA sequence is completely identical to the amino acid sequences near the N-terminal and C-terminal determined from the chum salmon growth hormone peptide, It has been confirmed that the cDNA encodes chum salmon growth hormone [Japanese Patent Application Nos. 59-134536 and 59-2.
13360].

Further, of pSGH8 and pSGH14, which are plasmids different in restriction enzyme cleavage site from this group, pSGH14 containing a longer cDNA which seems to have almost full length, and the whole nucleotide sequence of its translation region was converted to M13 phage. Sanger method used (Sanger et al., Proc. Natl. Acad. Sci.), USA, 74 , 54.
63 (1977): Amersham M13 Cloning Sequencing Handbook (cloning an
d sequencing handbook)). The sequence is shown in SEQ ID NO: 1.

From the comparison with the known chum salmon growth hormone in the nucleotide sequence of SEQ ID NO: 1, it is considered that the number of bases 1-66 codes the signal peptide and 67-630 codes the mature peptide of the chum salmon novel growth hormone. The 22 amino acid sequence of the signal peptide of the polypeptide encoded by the cDNA is completely identical to that of the polypeptide encoded by pSGH1, but in the mature peptide portion, it is shown in SEQ ID NO: 1 of 188. In the amino acid sequences, the 12 underlined amino acids differ. Also, even within the 40 amino acid sequence at the N-terminal determined from chum salmon growth hormone polypeptide, 5 amino acids are clearly different, and the cDNA contained in pSGH14 is
It is thought that it encodes a novel fish growth hormone that is different from the pSHG1 group. Escherichia coli containing the recombinant plasmid containing pSGH14 was Escherichia coli ESGH14 (FERM
BP-611) has been deposited with the Institute of Micro Engineering on September 20, 1984.

Example 5 Construction of recombinant plasmid encoding novel chum salmon growth hormone: (1) Construction of recombinant plasmid pSGHIIB9 encoding mature chum salmon growth hormone from pSGH14: containing DNA encoding novel chum salmon growth hormone Plasmid pSGH14
5 μg of 20 mM Tris-HCl (pH 7.5), 10 mM MgC
l _2, and dissolved in a solution 100μl containing 10 mM NaCl, restriction enzyme MboII (manufactured by New England Bio Labs Inc.)
10 units was added and the digestion reaction was performed at 37 ° C. for 3 hours. Then, adjust the NaCl concentration to 50 mM, and add PvuII
10 units was added and the digestion reaction was performed at 37 ° C. for 3 hours. After this reaction solution was subjected to polyacrylamide gel electrophoresis, DEAE was performed.
-The 108 bp DN corresponding to the vicinity of the N terminus by the paper method
About 0.05 μg of A fragment was obtained.

Next, 5 μg of pSGH14 was added to 20 mM Tris-HCl.
(PH 7.5), dissolved in 40 μl of a solution containing 10 mM MgCl ₂ and 50 mM NaCl (hereinafter abbreviated as “Y-50 buffer”), added 10 units each of PvuII and HindIII, and heated to 37 ° C.
The digestion reaction was performed for 3 hours. From the reaction solution by the LGT method, the C-terminal side of the growth hormone encoded by pSGH14,
About 0.5 μg of a DNA fragment of about 3.3 Kb containing the 3'-untranslated region and the vector portion was obtained.

On the other hand, a translation initiation codon ATG required for expression of the DNA encoding mature chum salmon growth hormone was added, and the following DNA linker was synthesized for the purpose of ligating the vector DNA with the above DNA.

[0046]

[Chemical 4]

First, single-stranded DNA, 14 mer and 9 mer, was subjected to the usual triester method [R. Crea et al .: Proceeding of the National Academia of Science (Proc. Natl. Acad. Sci.) USA., 75 , 57
65 (1978)]. 39 pmole of 14-mer and 9-mer single-stranded DNA was added to 50 mM Tris-HCl (pH 7.5).
, 10 mM MgCl ₂ , 10 mM dithiothreitol and 1 mM ATP were dissolved in 20 μl of the solution, 6 units of T4 polynucleotide kinase (Takara Shuzo) was added, and the mixture was incubated at 37 ° C.
The phosphorylation reaction was performed for 60 minutes.

MboII-PvuII fragment (108 bp) 0.08pmole, PvuII-HindIII fragment (about 3.7 Kb) derived from pSGH 14 obtained above
0.02 pmole was added to 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl ₂ , 10 mM dithiothreitol and 1 mM ATP
Dissolve in 30 μl of a solution containing
5 μl of phosphorylation reaction solution was added. Add T4DN to this mixture.
6 units of A ligase was added and the binding reaction was carried out at 4 ° C. for 18 hours.

Escherichia coli HB101 [Bolivar et al., Gene ( 2 , 75 (1977)] strain was transformed with the reaction solution to obtain Ap ^r colonies, and plasmid DNA was recovered from the colonies. The pSGHIIB9 shown in Fig. 4 was obtained.
The structure of GHIIB9 was confirmed by agarose gel electrophoresis after digestion with HindIII, XbaI, BglII and BamHI.

(2) Integration of pSGHIIB9 coding region for mature chum salmon growth hormone into expression vector pGEL1: 5 μg of pSGHIIB9 was dissolved in 40 μl of Y-50 buffer, and 10 units each of BamHI and HindIII were added, and the mixture was incubated at 37 ° C.
A time digestion reaction was performed. From the reaction solution by the LGT method,
About 0.1 μg of a DNA fragment of about 1200 bp encoding the entire mature chum salmon growth hormone was obtained.

Separately, 5 μg of pGEL1 was dissolved in 40 μl of Y-50 buffer, 10 units each of BamHI and HindIII were added, and digestion reaction was carried out at 37 ° C. for 3 hours. L from this reaction solution
Approximately 2.7 including tryptophan promoter by GT method
About 0.1 μg of Kb DNA fragment was obtained. PSGHIIB9 obtained above
HindIII-BamHI fragment (about 1200 bp) of 0.01 μg and pGEL
Of HindIII-BamHI fragment (about 2.7 Kb) of 0.015 μg
0 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10
Solution 3 containing mM dithiothreitol and 1 mM ATP
Dissolve in 0 μl, T4 DNA ligase (Takara Shuzo) 6
A unit was added and the binding reaction was performed at 4 ° C. for 18 hours.

Escherichia coli HB101 strain was transformed with the reaction solution to obtain Ap ^r colonies, and plasmid DNA was recovered from the colonies to obtain pSGHIIC2 shown in FIG. pS
The structure of GHIIC2 is EcoRI, HindIII, ClaI, BglII, BamHI
It was cut with and confirmed by agarose gel electrophoresis. pSGH
Escherichia coli containing IIB9 and pSGHIIC2
ESGHIIC2 FERM BP-708 and ESGHIIC B2 FERM BP-70
7 has been deposited with the Institute of Microtechnology.

Example 6 Production of novel chum salmon growth hormone peptide by Escherichia coli containing pSGHIIC2: Escherichia coli W3110strA strain (FERM BP-732) was transformed with the recombinant plasmid pSGHIIC2 obtained in Example 5 by a conventional method. The Ap ^r colonies obtained were 8 ml of MCG medium [0.6% Na ₂ HP.
O ₄ , 0.3% KH ₂ PO ₄ , 0.5% NaCl, 0.1% NH ₄ Cl, 0.
5% glucose, 0.5% casamino acid, 1 mM MgSO ₄ ,
4 μg / ml vitamin B ₁ , pH 7.2] was inoculated and cultured at 30 ° C. for 18 hours. The obtained culture solution was centrifuged at 8,000 rpm for 10 minutes to collect bacterial cells. The cells were suspended in Laemmli sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and stained with Coomassie Brilliant Blue to detect a polypeptide band at a site having a molecular weight of about 25,000. This band was absent when E. coli without the plasmid was used. As a result, pSGH
It was found that Escherichia coli carrying IIC2 produces a large amount of chum salmon growth hormone polypeptide.

[0054]

INDUSTRIAL APPLICABILITY 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. It can be used for mass production of peptides.

[0055]

[Sequence list]

SEQ ID NO: 1 Sequence length: 630 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Chum salmon Tissue type: Pituitary sequence Features of 1..66 S sig peptide 67..630 S mat peptide sequence ATG GGA CAA GTG TTT CTG CTG ATG CCA GTC TTA CTG GTC AGT TGT TTC 48 Met Gly Gln Val Phe Leu Leu Met Pro Val Leu Leu Val Ser Cys Phe -20 -15 -10 CTG AGT CAA GGG GCG GCG ATG GAA AAC CAA CGG CTC TTC AAC ATC GTG 96 Leu Ser Gln Gly Ala Ala Met Glu Asn Gln Arg Leu Phe Asn Ile Val -5 1 5 10 GTC AAC CGG GTG CAA CAC CTC CAC CTA TTG GCT CAG AAA ATG TTC AAC 144 Val Asn Arg Val Gln His Leu His Leu Leu Ala Gln Lys Met Phe Asn 15 20 25 GAC TTT GAA GGC ACC CTG TTG TCT GAT GAA CGC AGA CAG CTG AAC AAG 192 Asp Phe Glu Gly Thr Leu Leu Ser Asp Glu Arg Arg Gln Leu Asn Lys 30 35 40 ATA TTC CTG CTG GAC TTC TGT AAC TCT GAC TCC ATC GTG AGC CCC ATC 240 Ile Phe Leu Leu Asp Phe Cys Asn Ser Asp Ser Ile Val Ser Pro Ile 45 50 55 GAC A AG CAG GAG ACT CAG AAG AGT TCA GTC CTG AAG CTG CTC CAT ATC 288 Asp Lys Gln Glu Thr Gln Lys Ser Ser Val Leu Lys Leu Leu His Ile 60 65 70 TCT TTC CGC CTG ATT GAA TCC TGG GAG TAC CCT AGC CAG ACC CTG ACC 336 Ser Phe Arg Leu Ile Glu Ser Trp Glu Tyr Pro Ser Gln Thr Leu Thr 75 80 85 90 ATC TCC AAC AGC CTA ATG GTC AGA AAC TCC AAC CAG ATC TCT GAG AAG 384 Ile Ser Asn Ser Leu Met Val Arg Asn Ser Asn Gln Ile Ser Glu Lys 95 100 105 CTC AGC GAC CTC AAA GTG GGC ATC AAC CTG CTC ATC GAG GGG AGC CAG 432 Leu Ser Asp Leu Lys Val Gly Ile Asn Leu Leu Ile Glu Gly Ser Gln 110 115 120 GAA GGG GTA CTG AGC CTG GAT GAC AAT GAC TCT CAG CAT CTG CCC CCC 480 Glu Gly Val Leu Ser Leu Asp Asp Asn Asp Ser Gln His Leu Pro Pro 125 130 135 TAC GGG AAC TAC TAC CAG AAC CTG GGG GGC GAC GGC AAC GTC AGG AGG 528 Tyr Gly Asn Tyr Tyr Gln Asn Leu Gly Gly Asp Gly Asn Val Arg Arg 140 145 150 AAC TAC GAA CTG TTG GCC TGC TTC AAG AAG GAC ATG CAT AAG GTT GAG 576 Asn Tyr Glu Leu Leu Ala Cys Phe Lys Lys Asp Met His Lys Val Glu 155 160 165 1 70 ACC TAC CTG ACC GTC GCT AAG TGC AGG AAG TCA CTG GAG GCC AAC TGC 624 Thr Tyr Leu Thr Val Ala Lys Cys Arg Lys Ser Leu Glu Ala Asn Cys 175 180 185 ACTCTGTAA 633 ThrLeu

[Brief description of drawings]

FIG. 1 cDNA synthesis by the Okaya Marburg method and the DNA
It is a figure showing the outline of the construction process of the recombinant plasmid containing.

FIG. 2 is a diagram showing an outline of the construction process of a recombinant plasmid containing cDNA by the Okayama-Berg method.

FIG. 3 is a diagram showing a restriction enzyme map of cDNA contained in pSGH1, pSGH3, and pSGH14.

FIG. 4 is a diagram showing the construction process of recombinant plasmids pSGHIIB9 and pSGHIIC2.

Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location A61K 37/36 AER 8314-4C C07K 13/00 8619-4H C12P 21/02 C 8214-4B (C12N 1/21 (C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (5)

[Claims] 1. A D encoding a novel fish growth hormone polypeptide having the peptide sequence shown in SEQ ID NO: 1.
NA. 2. A D encoding a novel fish growth hormone polypeptide having the peptide sequence shown in SEQ ID NO: 1.
Recombinant DNA incorporating NA. 3. The recombinant DNA according to claim 2, which is designated as plasmid pSGH14. 4. A D encoding a novel fish growth hormone polypeptide having the peptide sequence set forth in SEQ ID NO: 1.
Bacteria containing recombinant DNA incorporating NA. 5. The bacterium according to claim 4, wherein the bacterium belongs to Escherichia coli.