JPH01104180A - Growth hormone gene of fishes, recombinant plasmid of said gene, microorganism containing said recombinant plasmid, production of fish growth hormone with said microorganism and promotion of growth of fishes by said growth hormone - Google Patents

Abstract

PURPOSE:To produce a large quantity of growth hormone of fishes at a low cost, by forming a recombinant plasmid containing a prescribed vector linked with a gene coding an amino acid sequence of a growth hormone polypeptide originated from a specific fish. CONSTITUTION:A DNA coding growth hormone of tuna is sectioned from a plasmid containing said DNA and is integrated into a vector DNA and the obtained recombinant DNA is introduced into a microorganism. The resultant transformant is cultured to produce and accumulate the tuna growth hormone polypeptide, which is separated from the cultured product. The plasmid containing the DNA coding tuna growth hormone is preferably pTTS339. The preferable vector DNA is pKK223-3, pKK233-2, etc.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides a gene encoding a fish growth hormone polypeptide, a recombinant plasmid incorporating the gene,
The present invention relates to a recombinant microorganism incorporating the gene, a method for producing a fish growth hormone polypeptide using the microorganism, and a method for promoting the growth of a wide range of fish using the growth hormone polypeptide.

(Prior art) The growth hormone of tuna is biosynthesized in the pituitary gland, and is produced by tilapia (S, N, Farmer et al.).

Gen, Comp, Endcrin, 30.91 (1
976) ), sturgeon (S, N, Farme
r et al., Endcrinology, 108377 (1
981)).

Carp (A, F, Cook etc., Gen, Comp, End
Growth hormone has been isolated from chum salmon (Japanese Patent Application No. 59-68670) and its properties have been clarified.

However, there are almost no reports regarding the isolation of genes encoding such fish growth hormone polypeptides. Only a case concerning the growth hormone polypeptide gene of chum salmon is reported in JP-A-61-15699.

[Problems to be Solved by the Invention] Fish growth hormone has the effect of promoting the growth of fish, and is an extremely useful future feed for cultivation and fishing. However, at present, supplying this hormone at low cost and in large quantities requires a large amount of the pituitary gland of fish, which is essentially an impossible problem.

The present inventors previously isolated growth hormone from the pituitary gland of tuna, clarified its properties and primary structure, and demonstrated that this growth hormone of tuna can be an excellent growth promoting factor for teleost fish. (Special application 1986-
No. 281147).

The present inventors further conducted research to develop a method for supplying fish growth hormone in large quantities at low cost, and completed the present invention.

(Means for Solving the Problems) The present inventors have investigated a method for obtaining fish growth hormone genes using recombinant DNA techniques. DNA encoding a fish growth hormone polypeptide;
A recombinant plasmid and microorganism containing this were produced.

The present invention has further advanced research and found that tuna growth hormone polypeptide is produced and accumulated in cultured bacterial cells by culturing a microorganism containing a plasmid incorporating DNA encoding tuna growth hormone. When a partially purified product was administered to tuna and sea bream, a growth-promoting effect was observed. Furthermore, the present invention has discovered that the amount of growth hormone polypeptide produced can be increased by linking two growth hormone structural genes.

The present invention will be explained in detail below.

Total RNA was extracted from the fresh pituitary gland of tuna, and it was extracted from oligo dT cellulose (oligo dT cellulose).
ose) column to prepare RNA (polyA-RNAΔ) having polyadenylic acid (polyA). Next, using this total poly A-RNA as a template, 2
Synthesize heavy chain DNA. Recombinant DNA is in vitro DNA
It can be easily obtained by inserting the synthetic double-stranded DNA prepared above into vector DNA such as E. coli plasmid DNA using A recombination technology.

Synthesis of cDNA and integration of cDNA into a vector were performed using the method of Okayama-Berg (Okayama & Berg).
Berg;Mo1. Ce11. Biol, 2,16
1. (1982) ) (see Figure 2).

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

First, vector primer psV7186 (
(manufactured by Pharmacia) in an appropriate solution, e.g. 50mM)
Lis/llCl (pH 7,5); MgCh (e.g. 8mM), calcium chloride (e.g. 30mM)
), dNTP (dATP, dGTP, dTTP, dC
TP, e.g. 2mM each) dithiothreitol (e.g. 1
mRNA solution (e.g., a small amount of 3mM Tris-
11cI (pH 7,5) solution] and reverse transcriptase (e.g. 100OU/ml)
[Manufactured by Takara Shuzo Co., Ltd.] was mixed and kept at a constant temperature (e.g. 37°C).
, Allow to react for a certain period of time (for example, 60 minutes). An oligo dC chain is added to the 3' end of the RNA-DNA duplex thus obtained.

First, a solution containing an RNA-DNA double strand is treated with phenol/chloroform, an equal volume of, for example, 4M sodium acetate is added, and ethanol precipitation is performed with twice the volume of ethanol.

The resulting pellet was dissolved in a small amount of sterile water to a final concentration of, e.g. 1mM cobal chloride)+dCTP (e.g. 70
u M), cacodylic acid (pH 6,8) (e.g. 1
40mM).

Tris-HCI (pH 6,8) (e.g. 30mM)
, dithiothreitol (for example, 0.1 mM) is added to the vector primer solution.

Terminal deoxynucleotidyl transferase is added to this reaction solution at a concentration of, for example, 1500 U/d, and maintained at a constant temperature (e.g. 37°C) for a period of time (e.g. 5-12) until 8-12 oligo dC chains are added. 10 minutes) Allow to react.

Next, the RNA-DNA double-stranded vector primer to which the oligo dC chain has been added is cleaved with old ndI [[. Tris H
CI (pH 7,5) (e.g. 10mM) 1MgCh
(e.g. 6mM) and NaCl (e.g. 60mM)
Add the vector primer to a solution consisting of old ndI
[(e.g. 20U) and heated at a constant temperature (e.g. 37°
C), Incubate for a certain period of time (eg 2 hours). The reaction product was extracted with phenol/chloroform and precipitated with ethanol to obtain a cDNA vector primer.

Add a linker to the (DNA) vector primer.
(psV1932) (manufactured by 7 Almasia),
Tris.)IcI (e.g. pH 7, 5, 20mM)
MgCh (e.g. 4 mM), (NH4)zsO
i (e.g. 10mM), KCI (e.g. 0.I
M) β-nicotinamide adenine dinucleotide (β-
NAD) (e.g. 0.1 mM) with E. coli DNA ligase (e.g. 6.7 μg/-) for a certain period of time (e.g. 15 hours) at a constant temperature (e.g. 12 °C).
).

Through this reaction, cDNA and linker DNA are circularized. Add dATP to this reaction solution.

Add dCTP, dTTP, and dGTP to a final concentration of 40 μH, and add β-NAD (e.g., final concentration of 0.15 m
M) E. coli DNA ligase (e.g. 4.4 μg/ml)
Ribonuclease H (e.g. IOU/rd) DNA
Add polymerase I (e.g. 17.7 U/ml),
By replacing the RNA portion with DNA, a recombinant plasmid containing complete double-stranded DNA is obtained.

The recombinant plasmid thus obtained was introduced into E. coli.

For example, Escherichia coli DNI strain was prepared using the method of Hanahan et al.
, 166557 (1983)). Transformants can be selected by ampicillin resistance as a marker.

The transformed Escherichia coli is immobilized on a nylon membrane and hybridized with a synthetic NA probe having a DNA sequence predicted from the known amino acid sequence of bluefin tuna growth hormone, and those that strongly form are selected (Pr
oc, Natl, Acad, Sci.

u, s, ^, 723961 (1975) Grunsfe
in-Hogress method).

Probe DNA was obtained using the triester method (J, Am, Chem
.

Sco, 977327 (1975)).

Selection with synthetic probes is performed by dot blotting, 5
The method of Other et al. (J, Mo1. Biol, 98
5 (13 (1975)), and by this method, recombinant plasmid DNA having a gene complementary to bluefin tuna growth hormone mRNA can be identified.

Production of tuna growth hormone polypeptide by expression of DNA encoding tuna growth hormone in microorganisms: Cutting out the DNA from a plasmid containing the DNA encoding tuna growth hormone, and integrating the DNA into vector DNA, resulting in a recombinant. Tuna growth hormone polypeptide can be produced by introducing DNA into a microorganism and culturing the resulting transformant to produce and accumulate tuna growth hormone polypeptide in a medium, and collecting it. D encodes tuna growth hormone
The above pTTS339 is a suitable example of a plasmid containing NA.

Any vector DNA can be used as long as the inserted DNA can be expressed in microorganisms, but preferably vector DNA is tac (trp-1
ac), trp+λPL, ara, recA promoters, etc., and Shine Dalgarno (
For the expression vector having the SD) sequence, vector DNA in which the SD sequence and the start codon (ATG) of the inserted gene have been adjusted to 6 to 20 bases is used. Specifically preferred vector D
Examples of NA include pKK223-3 and pKK233-2 (manufactured by Pharmacia Japan).

DNA encoding tuna growth hormone and vector DNA
A recombinant with A can be created using a general method such as restriction enzymes or related enzymes.

As a specific example, in the case of pTTS339, as shown in FIG. 4, first the NcoI-RsaI fragment encoding tuna growth hormone was obtained from pTTS339, and then
The digested material was subjected to low melting point agarose gel electrophoresis (La
rs Wieslander, Analytical
Biochemistry 9B+305 (1979)
) and polyacrylamide gel electrophoresis to determine the DN encoding the N-terminal side of tuna growth hormone polypeptide.
Obtain A fragment. The protruding part of this DdeI-digested fragment was filled in with Klenow enzyme to form a synthetic NA linker as shown below.
Create.

The above DNA fragment and a synthetic NA linker are ligated with T4 DNA ligase to create pNEs8s shown in FIG. Furthermore, a DNA fragment encoding tuna growth hormone (Ncol-RsaT fragment) was digested with C1al to obtain a fragment encoding the C-terminus (C1ar-Rsarl!Ji fragment).
was created, ligated with C1al-Pstl-digested pNEs8s using T4 DNA ligase, and after blunting the uncombined ends with T4 DNA polymerase, T4DNA was added again.
A recombinant plasmid (pTES8S) containing the gene DNA encoding tuna growth hormone without the signal portion was ligated with A ligase to create a recombinant plasmid (pTES8S). Furthermore, with the aim of increasing the expression of tuna growth hormone, the fifth
As shown in the figure, the replication origin of pKK223-3 was
c19 replication origin (Miki et al. + Pr
Otein Engineering, 327 (
1987)) Created the vector pUK I and
l-11ind I[I digested.

This and pTEs8s were digested with NcoI-HindllI. Full length DNA and T encoding tuna growth hormone
4 DNA ligase. Furthermore, the protruding ends were blunted with Klenow enzyme and ligated again with TDNA ligase (pUEs13). This was further digested with AatII-old ndlI to remove the untranslated region at the 3-end, and after blunting the ends with T4 DNA polymerase, T4 DNA
They were ligated using ligase to create pUEs13s.

This plasmid encodes mature bluefin tuna growth hormone.

The same process was performed to transform the tuna growth hormone gene into 2
It becomes possible to create a plasmid containing

For example, the above pUEs13s is converted into Bam1ll-Sal
After T-digestion, a fragment containing the promoter region, the region of the gene encoding growth hormone, and the terminator was recovered by low-melting-point agarose electrophoresis, and this fragment was ligated with pUEs13s that had been treated with Bam old, and then treated with Klcnow. , puES13S in which two tuna growth hormones were connected by ligation treatment again.
-2 can be created (see Figure 6).

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 50
μg of DNA to 1-150 InM (preferably 10-50 InM)
Tris-KCI (pH 6.0-9.0, preferably 7.0-8.5) 0-200mM NaCl or KCI, O-5(]oM (preferably 5-20mM)
) in a reaction solution containing 1 g of MgCl.
0 units (preferably 1 to 5 units per 1 gg DNA) at 10°C to 70°C for 10 minutes to 20 hours. The reaction is usually stopped by adding an equal amount of water-saturated phenol or
Restriction enzymes can be inactivated by heat treatment at 0 to 75°C for 810 to 30 minutes. The binding reaction of DNA fragments is carried out from 1 to
100mM (preferably 10-50mM) Tris IC
ICpH6.0-9.0. preferably 7.0 to 8.0)
1-50mM MgClg (preferably 5-20mM
) l-11-1O preferably 0.5-3mM) ATP,
T4 DNA ligase, 0.1-1000 mM (preferably 5-11) wM) in a reaction solution containing DTT.
The temperature is 1 to 30°C (preferably 4 to 16°C). The recombinant plasmid generated by the ligation reaction can be used, for example, by the transformation method of Lanahan et al.
: Journal of Molecular Bi
166557 (1983)) into E. coli. In order to isolate recombinant plasmid DNA from E. coli having the DNA, for example, the method of Birnboin et al.
cid Re5earch, 1513. (197
9)) Alternatively, the method of Holmes et al. [Holmes et al. + Analytical Biochemistry
114193)]. Plasmid DN
The cleavage site of restriction enzyme A is confirmed by using agarose gel electrophoresis or polyacrylamide gel electrophoresis after cleavage with the restriction enzyme. Furthermore, to determine the DNA base sequence, the method of Sanger et al. (Sanger et al., Procee
ding National Academy
5science USA, 745463 (1977
)), Maxam Gilbert, etc. (Maxam G11b
ert:Proceeding National A
academy of 5science USA 74
, 560 (1977)).

The bluefin tuna growth hormone peptide of the present invention can be produced as follows. Escherichia coli DHI is transformed using a plasmid (for example, pUEs13S), and pUEs13s-carrying bacteria are selected from ampicillin-resistant colonies. This transformed E. coli JM109/pUEs13s has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMB P-1807.

By culturing this in a nutrient medium, bluefin tuna growth hormone polypeptide can be produced in the culture. As the medium used here, any medium suitable for growth of E. coli and production of tuna growth hormone polypeptide can be used. Carbon sources include glucose, glycerol, fructose, lactose, sorbitol, etc.; nitrogen sources include meat extract, yeast extract, corn stave liquor, tryptone, casamino acids, (Nl14) z S(14. NH4Cl, etc.) However, as other nutritional sources, KH2PO41KH2P
O41NaJPO4+ NaHzPO4+ NaCl,
MgC1+ Mg5Oa, thiamine, thymine, etc. can be used. The culture is carried out at pH 6.0 to 8.5. Temperature 15°C
Culture is carried out at ~42°C with aerated agitation.

Since the bluefin tuna growth hormone polypeptide accumulates in the cultured cells after 1 to 24 hours of culture, the cells are collected from the culture, and the polypeptide is collected from the cells according to a conventional polypeptide extraction method.

To confirm the bluefin tuna growth hormone polypeptide in the bacterial cells, Laemmli sample buffer (Laemmli) was used.

Nature 227 680 (1970)) was directly mixed with bacterial cells, heated and dissolved, and subjected to 5OS-polyacrylamide gel electrophoresis (Laemmli, Nature No. u).
680 (1970)) and dyed with Coomassie Brilliant Blue R250 or G250 to confirm its production.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Preparation of polyA-RNA from the pituitary gland of bluefin tuna: The guanidium thiocyanate method (J
, R. Feramisco et al., J. Biol.
, 25711024 (1982)), polyA
Metsenger RNA (hereinafter abbreviated as mRNA) having the following was prepared as follows. 0.7 g of frozen pituitary gland of bluefin tuna was mixed with 4M guanidium thiocyanate, 0.5% sarcosine, 25mM sodium citrate (pH 7.0).
), 16 m of a solution containing 0.1% antiform and 0.1 M 2-mercaptoethanol! The mixture was crushed using a Teflon homogenizer. To this homogenate, O, l
16 m each of M sodium acetate, phenol and chloroform/isoamyl alcohol (29:1 v/v)
Proteins and nucleic acids were removed by adding 100% of the total amount of protein and treating at 60°C for 10 minutes, and then repeating the same treatment with half the amount of the previous treatment.

To the clear liquid thus obtained was added 3.6 ml of 2M potassium acetate, and then 80-20% ethanol was added to give a solution of -20%.
The total RNA was collected as a precipitate. Approximately 1.8 mg of the obtained RNA was mixed with 0.5 M NaCl,
It was dissolved in 10mM Tris.HCI (pH 7.5).

After incubation at 65°C for 10 min, total RNA was transferred to an oligo dT cellulose column (Collaborati
(manufactured by Re5earch Ink), and the mRNA having adsorbed polyA was treated with 101 Tris/llCl.
(pH 7.5) to obtain about 200 μg of polyA-containing mRNA.

Example 2 Synthesis of cDNA and insertion of the DNA into a vector: Oka
Yama-Berg's method (Mo1.Ce11.Bio
! +2 +16L (1982)) cDNA was synthesized and a recombinant plasmid incorporating it was constructed. An outline of the process is shown in Figure 2 of the attached document.

Plasmid ps with oligo dT, which is a vector primer
Approximately 2 μg of V7186 (manufactured by Pharmacia) and approximately 10 μg of polyARNA obtained in Example 1 were mixed with 50 mM l-Lis.tlCl (pH 8,3), 8 mM MgCh,
Dissolve in 30μ2 of a solution containing 30mM KCI and 1mM dithiothreitol, and add dATP and dCT to this solution.
P, dTTP, and dGTP were each added to a final concentration of 2 mM. Next, reverse transcriptase (hereinafter referred to as RT Ca5e
) at 42°C to give 1000U/d.
cDNA was synthesized by reacting for 00 minutes. After the reaction, the DNA recovered by ethanol precipitation was mixed with 30mM) Lis-HCI.
(pH 6,8), 140mM cacodylic acid (pH 6,8)
), terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT) was dissolved in a solution containing 1mM CoC1z and 70UM dCTP, and the mixture was incubated at 37°C.
The reaction was carried out for 9 minutes to add 8 to 12 dC strands to the 3'-end of the RNA-DNA duplex.

The reactant was mixed with phenol/chloroform (1:
lv/v), and the cDNA vector primer DNA with the dC chain added was recovered by ethanol precipitation. The resulting DNA was then treated with Tris-IICI (pH 7).
,5) , 6mM MgC1z and 60mM NaC
After dissolving in 40 μl of a solution consisting of +, 20 U of old nd
[[I (manufactured by Takara Shuzo ■)] was added and incubated at 37°C for 2 hours to cleave the old nd■ site.The reaction solution was treated with phenol/chloroform (1:1v/ν), and then precipitated with ethanol. cD with 1.6 μg of dC chain added
NA vector primer DNA was obtained. This DNA
Dissolve 1.6 μg in 9 μl of sterile distilled water, then add 45 μl
of 2M NaCl, containing 71 μl of 1mM EDTA 10
) Lis-HCI (pH 7,5) and 0. Iμg
/rIi oligo-dG linker-DNA psV193
2 (manufactured by Pharmacia) and heat-treated at 65°C for 5 minutes and then at 42°C for 60 minutes.
M) Lisu-HCI (pH 7,5), 4.0mM
MgC1z,IOIIIM(NH4)zsOn, o
, IM KCI, and 0.1 mM β-NAD to make the total volume 900 μl.

To this was added 6 μg of Escherichia coli DNA ligase (manufactured by Takara Shuzo), and the mixture was incubated at 12° C. overnight. Then a final concentration of 40 μM dNTPs ('dATP, dCTP.

dTTP, dGTP), 0.15mM
β-NAD, 4.4μg/- E. coli DNA ligase, IOU/mZ ribonuclease H, 18U/mfDN
Add A polymerase■ and heat to 12°C, 115°C, and 125°.
The reaction was carried out at C and room temperature for 300 minutes each. Circularization of recombinant DNA including cDNA and RNA-DNA in the above reaction
The RNA portion of the A double strand was replaced with DNA to obtain a complete double stranded DNA recombinant plasmid.

Example 3 Recombinant DNA containing bluefin tuna growth hormone cDNA
Selection: Using the recombinant plasmid obtained in Example 2, Escherichia coli strain DHI was purified by the method of Hanahan et al. (
D. Hanahan et al. J. Mo1. Biol, plate 557 (1983)]. Of the approximately 20,000 obtained, 5,000 were identified on the nylon membrane.

In this transformed strain, a synthesized RNA corresponding to the 67th to 71st amino acid sequence from the N-terminal side of bluefin tuna growth hormone, i.e., 5'-GACTTCTGTAACTC
Eleven transformants were selected that strongly hybridized with T-3' at 42°C. The 11 strains thus obtained were C.
It also strongly hybridized with a synthetic NA probe corresponding to the amino acid sequence near the end, namely 5-^^GGACATGCACAAG-3'. Recombinant DNA held by 11 transformants of 2nd class was transformed into PTTS 1 and PTTS
2-1. PTTS3.

PTTS4, PTTS6, PTTS7, P
TTS24. PTTS37. PTTS39, PT
They were named TS339 and PTTS 2-5. Both are D predicted from the amino acid sequence of bluefin tuna growth hormone.
Since it has an NA sequence, it was thought to contain cDNA encoding growth hormone polypeptide.

Example 4 Creation of bluefin tuna growth hormone gene map using restriction enzymes: Various genes obtained in Example 3 (PTTSI, 2-1.
3゜4, 6. Restriction enzyme maps of 7.24.37.39.339 and 2-5) were created. Shown in Figure 1.

Example 5 Base sequence of the plasmid PTTS339: The entire base sequence of the inserted DNA (bluefin tuna growth hormone gene) including the translated region and untranslated region of PTTS339 of the plasmid obtained in Example 4 was determined. The base sequence was determined using the Dideoxy method (F, Sanger + 5cie
nce, 214.1205-1210 (1981)
) was used. The arrangement is shown in FIG. 1-51 in the figure
encodes the signal peptide, and 52-612 encodes the mature peptide of bluefin tuna growth hormone. The N-terminal boolobes used in Example 3 exist at 199-213, and 15
Thirteen of them matched, and the C-terminal probe was 538-
There were 552 items, and all 15 items matched. PTTS2-
5. E. coli containing PTTS24 (E, col, respectively)
i C600PTTS2-5゜E, coli DHI
, PTTS24) is FER dated May 12, 1986.
? It has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as IP-9367 and 9368.

Example 6 Creation of recombinant plasmid pUEs13s encoding adult 7p% bluefin tuna growth hormone: Plasmid pTTS33910ttg containing DNA encoding bluefin tuna growth hormone was added to 10mM)
Dissolve in 50μ2 of a solution containing HCI (pH 8,0), 10mM MgCIz and 80mM NaCl, and add the restriction enzyme NcoT-RsaI-Hind [(manufactured by Zenshuzo ■) 2
0 unit was added and the digestion reaction was carried out at 37°C for 16 hours. From this reaction solution, 2 μg of a fragment (770 bp) encoding bluefin tuna growth hormone peptide was obtained by low melting point agarose gel electrophoresis.

Next, this NcoI-RsaI fragment (770bp)
2 ug 10mM) Lisu-HCI (pH 7,5)
, 7mM MgCIz, 150mM NaCl.

Dissolve in 30 μl of 7mM 2-mercaptoethanol,
Ddel digestion was carried out at 7°C for 6 hours. This reaction solution was again subjected to low melting point agarose gel electrophoresis, and N
A 450 bp DNA fragment encoding the terminal end was recovered.

Next, in order to blunt the ends of this DNA, 25u1. 67mM potassium phosphate (pH 7.4), 6.7m
It was dissolved in a solution containing MMgCIz, 1 mM 2-mercaptoethanol, and 33 μm dNTP, and treated with 2 to 5 units of Klenow enzyme (manufactured by Zenshuzo ■). Next, the following DNA linker was synthesized for the purpose of adding a translation initiation codon ATG necessary for expression of this blunted 450 bp DNA fragment. [Manufactured by Zenshuzo ■] This phosphorylated synthetic linker and 450bp DNA fragment were combined into 20u
6611IM Tris HCl (pH 7,6),
66n+MMgCh, 10mM DTT, 0.1mM
It was dissolved in a solution containing ATP (hereinafter referred to as ligase buffer) and reacted with 600 units of 74 DNA at 16°C for 8 hours. After precipitating this synthetic linker-bound fragment with ethanol, 20 μl of 10 mM) Lis-HCI (pH
8,5), 7mM MgC1z.

Dissolve in 80mM NaCl solution (hereinafter referred to as Nco I buffer), add 4 units of NcoI (manufactured by Takara Shuzo), and add 37
°C for 2 hours. This fragment was treated with Ncol-Pstl in NcoI buffer.0, 5μg pKK23
3-2. Mix in gauze buffer and add 300 units of T4.
A ligation reaction was performed using DNA ligase at 16°C for 58 hours. Furthermore, for the purpose of smoothing unbound portions, the reaction solution was precipitated with ethanol, and then 20 μl of 67 mM) Lis-○CI (pH 8,8) was added.

6.7mM MgCb, 16.6mM (NH41zS
On, 10n+M 2-mercaptoethanol, 6.
7mM EDTA, 330μM dNTP (dA
TP, dTTP, dCTP, dGTP) (hereinafter referred to as T4 polymerase buffer), and
React for 30 minutes with 74 units of DNA polymerase,
After the reaction, add ATP to a final concentration of 1 mM, and add 300
One unit of T4 DNA ligase was added and the reaction was carried out at 16°C for 116 hours to circularize (pNES8S). This circularized pNES8S was
After transforming Escherichia coli JM109 and examining the ampicillin-resistant strain, the recombinant plasmid was isolated according to the method of Birnboim et al., and its orientation was examined using various restriction enzymes. ) was obtained. Next, 2 ug of the Ncol-Rsal fragment (770 bp) encoding the tuna growth hormone polypeptide extracted from pTTS339 was mixed with 20 ul of 10 mM Tris-IIcI (pH 8,0), 7 mM MgC.
In a solution containing 1g, 50mM NaCl (hereinafter referred to as C1a
I buffer), digested with 10 units of C1al, and subjected to low melting point agarose gel electrophoresis to recover a 490 bp DNA fragment encoding the C-terminal side. to this,
0.5 μg of pNEs8s was treated with 10 units each of CIaI and Pstr in 20 μfcial buffer, dissolved in gauze buffer, and 300 units of T4DN was added.
A ligase treatment was performed at 16°C for 8 hours. After ethanol precipitation to remove unbound sites, T4 polymerase buffer 2
Dissolve in 0 μl and add 4 units of T4 DNA polymerase to
A smoothing reaction was carried out for 115 minutes at 0''C.Furthermore, ATP was added to this reaction solution to a final concentration of 1mM, and T4 was added again.
300 units of DNA ligase was added to circularize. This was transformed into E. coli JM109 in the same manner as above, ampicillin-resistant bacteria were selected, and a recombinant plasmid was isolated from the bacteria. (pTE
S8S) retention bacteria were obtained (see Figure 4).

Next, the replication origin of this recombinant plasmid is the colicin EI system, and it is known that there are 20 to 50 replication origins per E. coli cell. Recently, this origin of replication was converted to pUc.
system plasmid (200-50 per E. coli cell)
A method has been established to increase the copy number by replacing the 0 copies. (Miki et al., Protein Engineer
ing, 327. (1987)) Therefore, the same process was used for zero reports to detect an increase in copy number.

Details are given below. pKK223-3 (manufactured by Pharmacia Japan), 2 μg and pUc19 (manufactured by Takara Shuzo ■) 2
μgwosoh('11.20u l (7) IOIIIM
To'Jsu・f((:I(pH8,0), 150mM
2-Mercaptoethanol was dissolved in a solution containing 0.1 mM EDTA and digested with 1z units of Pvul. After ethanol precipitation, further added 20μ2 each of 10mM Tris-HCI (pH 7,5), 50mM KCI,
10mM 2-mercaptoethanol, 0.1mM
1z units of Pvu I dissolved in a solution containing EDTA
Digested with I. Through these treatments, a fragment that does not contain the replication origin of pKK223-3 and a fragment that contains the replication origin of pUc19 were extracted by low melting point agarose gel electrophoresis, and a 20μ! Dissolve in glue gauze buffer and treat with 300 units of 74 DNA ligase for 16
'C, I did it for 5 hours. As a result, a plasmid pUKI having the replication origin of ptlc19 was obtained. pUK I was transformed into Escherichia coli JM109 using the method described above to obtain cells containing the desired plasmid. This bacterial body (pUK I /
2 μg of pU isolated from JM109) using the method described above.
K1 was added to 20 μl of 10 mM Tris-HCl (pH 7).
,5), 7+wM MgC2z, 60mM Na
A solution of C1 (hereinafter, dissolved in Hindl [[[buffer solution]]
Each was digested with 10 units of 3HaI-Hind m. This and 3 μg of the aforementioned pTEs8s were dissolved in Ncol buffer and 10 units of NcoI-Hi was added to each.
A 720 bp fragment encoding mature tuna growth hormone was obtained by digestion with nd m. This fragment was digested with 10 units of SmaI-HindI [[
11 μg of UK is dissolved in ligase buffer and ligated with 300 units of T4 DNA ligase at 11° C. for 8 hours. In order to blunt the ends, which are unbound sites, this solution was precipitated with ethanol, dissolved in 25 μl of Klenow buffer, 2 units of Klenow enzyme was added, and reacted at 30° C. for 15 minutes.

This reaction solution was again precipitated with ethanol, and 20 μm of ethanol was precipitated. It was dissolved in glue gauze buffer and circularized with 300 units of T4 DNA ligase. This was transformed into E. coli JM109 as described above, and the desired recombinant plasmid (pUEs13) was obtained.
We obtained a strain with

Furthermore, a recombinant plasmid was isolated from this bacterial cell, and 1 μg of it was added to 20 μl of old ndl [37 μg after buffer digestion].
Digested with old ndlII and AatII for 6 hours at °C.

After ethanol precipitation, this was dissolved in 20 μl T4 polymerase buffer and treated with 5 units of 74 DNA polymerase for 3
After smoothing the ends for 0 minutes at 30"C,
Add 300 units of T to give a concentration of mMATP.
4 DNA ligase was added, and the mixture was reacted at 16° C. for 8 hours to circularize. This was transformed into Escherichia coli JM109 as described above to obtain a strain containing the desired recombinant plasmid (pUEs13s) (see Figure 5).

The structure of pUEs13s is Ncol, C1aI, l
1ind m + AatU + EcoRI, ,
It was cleaved with Sma I and confirmed by agarose gel electrophoresis. The base sequence near the N-terminus of the DNA encoding mature tuna growth hormone in pUEs13s was confirmed to be the following sequence according to the method of Sanger et al.

As a result, it was found that pUEs13s contains DNA encoding mature tuna growth hormone polypeptide.

Example 7 Recombinant plasmid connecting two genes encoding mature bluefin tuna growth hormone: pUEs13s (2 ug to 20 uA of 10 mM) squirrel, HCI (pH 8,0), 7 uM MgC1,
, 100mM NaCl, 2mM 2-mercaptoethanol, then 5 units of BamHI
was added and digestion was performed at 37°C for 4 hours.

After ethanol precipitation, add half of it to 20ul of 10mM)
Lis HCl (pH 7,5), 7mM MgCh,
It was dissolved in 125mM NaCl, 7mM 2-mercaptoethanol solution and digested with 4 units of Sea I. This was subjected to low melting point agarose gel electrophoresis to obtain a fragment (approximately 1700 bp) containing the promoter region, DNA encoding mature tuna growth hormone, and transcription termination sequence.
) and the remaining half of the BamHI digested with 300 units of T4 DNA ligase in a ligase buffer to form a recombinant plasmid containing the tuna growth hormone gene in tandem as shown in Figure 6. (pUEs1
3s-2) was created. E. coli JM109 after similar treatment
transformed into. This transformed colon surface J old 09/p
UES13S-2 was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the FERMB P-1799.
It has been deposited as.

Example 8 Production of tuna growth hormone polypeptide by Escherichia coli containing pUas13s and ptlEs13s-2: Escherichia coli Jl'1109 carrying the recombinant plasmids ptlEs13S and pUEs13s-2 obtained in Example 6.8 was treated with 8% tryptone and 5% tryptone. % Yeast Extract, 5%N
The cells were cultured in 10mf YT medium containing aC1 (pH 7,2) until 37'CO,0 was 0.7. O,D=0.7
isopropyl β-D- to a final concentration of 0.1 mM.
Add thiogalactopyranoside (IPTC;) and
Cultured for hours.

After collecting 1 mf of the bacteria, they were suspended in Laemmli's sample buffer, dissolved by heating, and subjected to 5DS-polyacrylamide gel electrophoresis. After electrophoresis, it was stained with Coomassie brilliant blue and decolorized, and the molecular weight was 21.000~
A polypeptide band was found at the 22,000 site. This electrophoresed gel was subjected to Western blotting (Beisiegel et al., Journal of B
iological chemistry+ 257+
13150), it was found that tuna growth hormone was produced in large quantities because it bound with an anti-tuna growth hormone antibody.

Example 9 Examination of expression efficiency of tuna growth hormone gene and productivity in various hosts based on the distance from the SD sequence to the start codon ATG: In addition to pUEs13s created in Example 6, the distance from the SD sequence to the start codon ATC was A similar modified vector was created. (p[lEs13s and pUEs1
In 3S-2, the distance between SD and ATG is 13 nucleotides. ) Plasmids other than those described above were created in which the distances from the SD sequence (AGGA) to the start codon (ATG) were 10, 12, 14, and 15, respectively. The creation method is described below. First of all, 10 things are 1
μg of pUKI was dissolved in 48μ2 of old ndl[I buffer, added to 1μ2 each of EcoR [10 units] and 1μg of pUKI.
Add β Hind m (10 units) and incubate at 37°C for 6
Time reacts. After ethanol precipitation, this and 0.5 ug of NcoI obtained in Example 6, old ndI [[digested 7
The 20 bp fragment encoding mature tuna growth hormone is dissolved in ligase buffer and ligated with 300 units of T4 DNA ligase at 16°C for 8 hours. In order to blunt the ends, which are unbound portions, the same treatment as in Example 6 was performed and recircularization was performed with T4 DNA ligase (pUEslo).
. 3-Untranslated region of this was removed by the same process as Example 6,
pUEslos was created using the same process. Twelve pieces were created as follows. 1 μg of pUc9 (Mik
i et al., Protin Engineering Sat., 3
27 (1987)) in 10mM Tris-HCI (pH
7,5), 7mM MgC1z, 175mMNa
C1,7mM 2-mercaptoethanol, 0.2mM
After dissolving in 18 μl of EDTA solution, cut with 12 units of 5alI. This was precipitated with ethanol, further dissolved in 18 μl of old nd■ buffer, and then recut with 10 units of old ndnl.

After ethanol precipitation, the above-mentioned 720 bp growth hormone gene is ligated in the same manner, and after being similarly treated with Klenow enzyme, it is religated (pNS2). This reaction solution was then ethanol precipitated, dissolved in 18 μl of old ndl[r buffer, and added with 10 units each of EcoRI and Hin.
After digestion with dI at 37°C for 5 hours and electrophoresis on a low melting point agarose gel, a gene fragment (small fragment, approximately 720 bp) encoding growth hormone is recovered. Furthermore,
To this fragment, 1 μg of pUK r was added to Ec in the same buffer.
The oRI, l1ind m-cleaved fragment was dissolved in 18 ul of ligase buffer and reacted with 300 units of T4 DNA ligase at 16°C for 8 hours to perform a ligation reaction.

As a result, pUEs12 can be created in which the distance between the SD sequence and the start codon ATG is 12 nucleotides. In addition, by the same process as pUEslo, pUEs12s
can be created. The 14 creation methods are as follows.

1 μg of pUKI was added as described in Example 6 or above.
and cut with EcoRE. This was subjected to low melting point agarose gel electrophoresis, a large fragment was cut out, and pSma I
Linker (manufactured by Zenshuzo ■) pGCCCGGGC50ng
was ligated in the same reaction as above to obtain pUKsl. This p
UKsl (2 μg) is again digested with Sma 1-Hindll using the same buffer and reaction time as above. After this was subjected to low melting point agarose gel electrophoresis again, a large fragment was cut out and a 720 bp growth hormone gene fragment (Nco T
-11indIII fragment) and ligase in the same manner as above, and after circularization in the same manner, pUEs14
get. pUEs14s is obtained in the same manner as above. 15
The individual items are as follows. 1 μg of pUKI was treated with Sma I-EcoRI in the same manner, and a large fragment was excised in the same manner. To this, pclal linker [Zen Shuzo ■
Co., Ltd.) pCATCGATG 50ng and a binding reaction,
Obtain pUKcI. This pUKcI (2ug) was
Dissolved in 18 μi of I buffer, each containing 10 units of C
Ial+tlindn [, ligated with the above 720 bp fragment, and circularized. This yields pUEs15, and similar processing yields pUEs15s. Next, pUEslos and pUE obtained by these processes are
s12s, pUEs13s, ptLEs145,
pUEs15s (nucleotide sequence was confirmed according to the method of Sanger et al., see Figure 7) was transformed into various Escherichia coli D111, HBIOI, R by the transformation method of Hanahan et al.
B791°C600, JM1 (13. JM105.
Transform into JM109.

Using this, an induction experiment similar to Example 8 was conducted and the results are shown in Table 1. The induction time was 6 hours after addition of IPTG, and the temperature was 37°C. The electrophoresed gel was subjected to densitometer to examine the expression efficiency.

(Margins below this page) Example 10 Examination of the biological activity of tuna growth hormone produced from Escherichia coli: In the same manner as in Example 9, bacterial cells cultured up 30 m of YT medium were collected, washed, and then disrupted by ultrasonication. The precipitate obtained was mixed with 2% Triton X-
After centrifugation, the precipitate was dialyzed overnight in 10 mM Tris-HCl (pH 7,0) containing 7 M urea, 1% 2-mercaptoethanol at 4°C.

This solution was mixed again with 101 Tris.HCI (pH 7.0
) After 1 night of dialysis at 40"C, it was subjected to affinity ramming with 1gG of tuna growth hormone as a ligand three times, and approximately 6
mg of tuna growth hormone was obtained. This protein is 5
OS-polyacrylamide electrophoretically single.

Using this, we conducted the following study.

Bluefin tuna fry 3 with body length of 15cm and body weight of around 100g
Thirty yellowfin tuna fry each with a body length of 13 cm and a weight of around 80 g were divided into two groups and placed in four sets of aquariums at a water temperature of 25°C. Similarly, 15 sea bream fry with a body length of 12 cm and a weight of around 40 g were placed in two sets of aquariums at a water temperature of 12°C.

Seawater was constantly circulated and dirt was filtered out. Food was given twice a day at 1 o'clock in the morning and at 5 p.m. until satiation. Add 0.5 tuna growth hormone obtained above to one set of each fish species four times every 5 days.
μg/100μ! Physiological saline was injected intraperitoneally. On the other hand, the remaining set was given 100 µm of physiological saline four times every 5 days to serve as a control. Figure 8 shows bluefin tuna, Figure 9 shows yellowfin tuna, and Figure 10 shows sea bream tuna, and the increases in body length and weight were examined every five days.

For reference, FIG. 11 shows the results of an experiment conducted on sea bream using tuna growth hormone purified from the pituitary gland of bluefin tuna.

From the above results, it was revealed that the tuna growth hormone polypeptide produced by E. coli has the same growth promoting effect as purified tuna growth hormone.

Escherichia coli containing ρUES13S and pUEs13s-2 (
pUEs 13S/Escherichia respectively
coli JM109+ pUEs 13S-2/Es
cherichia coli JM109) are FERMBP-1807 and FERM BP-1 dated March 22, 1988 and March 16, 1988, respectively.
799 and has been internationally deposited and entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology.

〔Effect of the invention〕

According to the present invention, fish growth hormone polypeptide can be produced in large quantities using microorganisms. It has been found that the produced growth hormone has a growth-promoting effect not only on tuna but also on sea bream, and it is hoped that it will have a growth-promoting effect on a wide range of fish species.

Furthermore, by linking two growth hormone structural genes, it was possible to increase the productivity of growth hormone polypeptide.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme map of the bluefin tuna growth hormone gene using restriction enzymes, and FIG. 2 shows an outline of the synthesis of cDNA by the Okayama-Berg method and the construction of a recombinant plasmid containing the DNA. Figure 3 shows plasmid PTTS
This is the complete base sequence of the tuna growth hormone gene in No. 339. Figures 4 and 5 show a method for preparing recombinant DNA from a growth hormone structural gene and a vector. Figure 6 shows a method for preparing a recombinant plasmid containing two consecutive growth hormone structural genes. The seventh tooth is pUEslos, p[I
Hs12s, pUEs13s, pUEs14s
Figures 8 to 10 show the changes in body weight and body length when the growth hormone obtained in the present invention was administered to juvenile bluefin tuna, yellowfin tuna, and sea bream, and Figure 11 shows the brain sequence of bluefin tuna, pUEs15s. The results are shown when growth hormone purified from the pituitary gland was similarly administered to young sea bream fish.

Claims (14)

[Claims] (1) A gene encoding the amino acid sequence of the fish-derived growth hormone polypeptide shown in FIG. (2) A gene encoding the amino acid sequence of a fish growth hormone polypeptide, which is a combination of two genes encoding the amino acid sequence of a fish growth hormone polypeptide shown in FIG. (3) The gene according to claim 1 or 2, wherein the fish is a perch. (4) The gene according to claim 1, 2 or 3, wherein the sea bass is tuna. (5) A recombinant plasmid obtained by linking the gene according to claim 1, 2, 3, or 4 with a desired vector. (6) Recombinant plasmid is pTTS339, pTTS
6. The recombinant plasmid according to claim 5, which is 2-5. (7) The host microorganism is the recombinant DN according to claim 5 or 6.
A transformant microorganism transformed with A. (8) The transformed microorganism according to claim 7, wherein the host microorganism is a microorganism belonging to the genus Escherichia. (9) A method for producing a fish growth hormone, which comprises culturing the transformed microorganism according to claim 7 or 8 in a medium, and collecting the fish growth hormone from the culture. (10) The method for producing a fish growth hormone according to claim 9, wherein the fish is a sea bass. (11) The method for producing growth hormone according to claim 10, wherein the sea bass is tuna. (12) A method for promoting growth by administering fish growth hormone obtained by the method according to claim 9 to fish. (13) The method for promoting growth according to claim 12, wherein the fish is a perch. (14) The method for promoting growth according to claim 13, wherein the sea bass is tuna.