JPH02219576A - Fish growth hormone gene - Google Patents

Abstract

PURPOSE:To provide the subject gene consisting of a fish growth hormone gene capable of coding an amino acid sequence containing a specified fish growth hormone polypeptide and enabling ready mass production of a growth hormone of a red sea-bream in a high state of purity using the gene recombination technique. CONSTITUTION:A fish growth hormone gene capable of coding an amino acid sequence containing a fish growth hormone polypeptide represented by the formula. The above-mentioned gene can be produced, e.g. by preparing a poly(A)RNA from the hypophysis of a red sea-bream which is cells producing a growth hormone thereof, synthesizing cDNA using the obtained poly(A)RNA as a template, coupling the resultant cDNA with a suitable vector, amplifying the coupled cDNA in a host cell, selecting a clone having the objective cDNA of the red sea-bream GH and carrying out isolation using the vector contained in the selected clone, or chemically synthesizing the objective acid based on the DNA sequence of the gene using the phosphoamidite method, etc.

Description

[Detailed Description of the Invention] Ryoto J. 2 Ko 1 The present invention provides a new fish growth hormone gene, more specifically a red sea bream (Pagrus major) growth hormone ((3rowth (ormone: (3 days)).
The present invention relates to a DNA sequence encoding a polypeptide, a vector containing the same, and a host cell harboring the vector.

Asahi - Beiichigo - ■ Growth hormone (Gl) is a polypeptide that is secreted from the pituitary gland of humans and other various animals and exerts a growth-promoting effect in these animals. It is well known that abnormalities in secretion cause diseases such as dwarfism and acromegaly [Kazuo Kusame, edited by Miura and Kusame, "Polypeptide Hormones", Asakura Shoten, pp. 177-191 (
1973)].

G)J extracted from the human pituitary gland has been clinically used to treat dwarfism caused by GH deficiency or secretion dysfunction, but its supply is extremely small and insufficient. It is insufficient to carry out proper treatment [Chain, Journal of the Japanese Endocrine Society, 60゜1492-1502 (198
4)).

On the other hand, GH from second-class animals is highly expected to be useful in terms of growth promotion and meat increase in livestock, fish, etc., but for the same reasons as above, research on G)l in such applications Development is hardly progressing.

In addition, GH is highly species-specific (and it is said that it is difficult to repurpose GH derived from a different species [see the above-mentioned document (1973)].
), research on GH has not progressed from this aspect either.

The first report on the chemical structure of GH was by Lee et al.
, H, Li et al.) (J, A
mer,Chem,soc,,88.2050(196
6)) Then, from gene isolation and structural analysis technology,
Gel of various animals, such as human Gel (J, M, M
artial et al., 5science, 20
5°602 (1979) L Rat GH (P, H.

5eeburg et at, Nature,
270.486 (1977)), bovine G) f and pig G
)l (P, )l.

Seebrug et al., DNA, 2.37
(1983)), Sheep GH (JP-A-60-1155
7@Ko! /Ii), chicken Gel and turkey GH (Special Publication No. Sho 59-501852), chum salmon G
H(3ekine et al., Pr0C, Nat
j, ACad, 3Ci,.

USA, 82, 4306 (1985), Eel G)
-J (Japanese Unexamined Patent Application Publication No. 62-22-22BOO@publication) etc., the structures of various G)l genes were also revealed, and using the isolated secondary genes, Various studies are being conducted on the production of GH using recombinant techniques, and studies on the practicality of the crude recombinant GH obtained using recombinant techniques have also been reported (K.

Takano et al., Endocrinal
, Japan, 5°589 (1986), etc.).

If the various GHs mentioned above can be applied to farmed fish as food resources, especially to major marine farmed fish such as red sea bream, yellowtail, and flounder,
Although its practical value is very high, when considering the species-specificity of Gel, the above research results on various mammals G)l are of no use at all. Reports on fish such as chum salmon and eel also have a large taxonomic gap between them and red sea bream, and their applicability is extremely low. Therefore, there is currently a strong desire in the industry to analyze the structure of G)-1 of the main marine cultured fish, which grows particularly slowly, and to establish a technology for its mass production.

Problems to be Solved by Akira The purpose of the present invention is to solve the above-mentioned red sea bream G.
The object of the present invention is to establish a new technology that makes it possible to easily produce H with high purity and in large quantities using genetic recombination technology, and to provide a red sea bream G)-1 gene that is particularly useful for this technology.

Means for Solving the Four Problems As a result of intensive research for the above-mentioned purposes, the present inventors have discovered a gene (DNA) encoding the amino acid sequence of red sea bream G)I useful for producing red sea bream GH using genetic recombination technology. Sequence) was successfully isolated and purified for the first time, leading to the completion of the present invention.

That is, the present invention relates to a fish growth hormone gene encoding an amino acid sequence containing a fish growth hormone polypeptide represented by the following formula (1).

Gln-Pro-I 1e-dinghr-ASI)-GIV
-Glrl-ArO-LeU-1'ge-Ser-I
1e-Ala-Val-3er-Arg-Val-Gl
n-old 5-teu-old 5-Leu-Leu-Ala-G
ln-ArQ-Leu-Phe-3er-Asp-Ph
e-Glu-3er-3er-Leu-Gln-Thr
-Glu-Glu-a? n-Leu-Lys-Leu-
Asn-Lys-I 1e-Phe-Pro-Asp-
Bge-Cys-Asn-3er-Asp-Tyr-I
Ie-11e-3er-Pro-¥? e-Asp-L
ys-)fis-Glu-Thr-Gln-Arg-3
er-3er-Val-Leu-Lys-Leu-Le
u-3er-I 1e-3er-■yr-Arg-te
u-Val-Glu-3er-■rp-Glu-Phe
-Pro-3er-Arg-3er-1(i)O Leu-3er-Gly-Gly-3er-Ala-P
ro-Arg-Asn-G n-GIy-11e-Hi
s-LeU-LeU-11e-Arp-Ala-ASn
-aig-Glu-Ala-ASn-CyS-Dinghr-
Leu (1) In this specification, peptides and amino acids, nuclear si groups, DNA sequences, and other abbreviations are expressed as IUPAC-IL
It follows the JB regulations or common symbols in the field.

Also, amino M in the polypeptides described herein
The numbers are indicated with Gin at the amino terminus (N terminus) of the red sea bream GH amino acid sequence shown in formula (1) above being 1, and counting sequentially in the direction of the carboxy terminus 1 m (C terminus).

The gene of the present invention makes it possible to easily produce red sea bream GH in large quantities through genetic recombination technology, and the red sea bream GH obtained thereby is extremely valuable in the fish aquaculture industry. be.

At present, the amino acid sequences of GH derived from various animals are known, but that of red sea bream G) has not yet been reported, and the amino acid sequence of red sea bream GH clarified by the present invention is different from that of any known GH. Incidentally, the amino acid sequence of the red sea bream GH of the present invention is only 3 different from human G).
It has only 3% homology, approximately 68% homology with chum salmon GH, and approximately 45% homology with eel GH.

The genes of the present invention will be described in detail below.

More specifically, the gene of the present invention includes a DNA sequence encoding the amino acid sequence of the fish growth hormone polypeptide represented by the above formula (1).

A specific example of the above DNA sequence is as shown in the following formula (4).

5°-CAG CCG ATG ACA GA
Ding GGCCAG CGT CTG T Ding C Ding C
CATG GC Ding GTCAGCAGA G Ding T
CAA CACC Ding CCACCTCCTCGC Ding
CAG AGA CTCT TC GACG
AG GCCAAACTGCACCCTG -3°
(4) Therefore, the gene of the present invention encodes the amino acid sequence of fish growth hormone (red sea bream GH), and a substance obtained by genetic engineering using this gene can exhibit the activity of red sea bream GH. As long as it is, the DNA sequence is not limited to the one shown in formula (4) above. For example, for each amino acid constituting the amino acid sequence of formula (1) above, a modified DNA sequence constructed by selecting an arbitrary codon from among the 1 to 6 known types of DNA codons may also be used. is included in the gene of the present invention as long as it enables the expression of red sea bream Gel activity. Also, based on the DNA sequence shown in formula (4) above, a DNA with ATG, which is a Met codon, added to its 5' end.
DNA sequences in which a stop codon such as TAA, TAG, TGA, etc. is added to the 3' end of a DNA sequence such as the A sequence are also included in the gene of the present invention.

Furthermore, the amino acid sequence of red sea bream GH is
Host cells obtained by creating a red sea bream GH expression vector by genetic recombination technology and integrating the vector are not limited to those represented by formula (1) as long as they exhibit the activity of As long as the red sea bream GH can actually be expressed, any part thereof, such as a part of the amino acid sequence at the N-terminus or C-terminus, may be deleted or substituted with other amino acid sequences, or It may be modified by further adding an arbitrary amino acid sequence to these. DNA sequences encoding second-modified amino acid sequences are also included in the genes of the present invention. As a specific example, poly(A>RN/'!- is prepared from a DNA sequence encoding a precursor of red sea bream GH, which will be described in detail in the Examples below, or from the red sea bream pituitary gland, which is a cell that produces red sea bream GH, and The DNA sequence of CDNA synthesized as a template can be exemplified.

The gene of the present invention can be produced by various methods shown below. As the method, for example, the above-mentioned red sea bream GH
Poly(A>RN
Wall A, use this as a template to synthesize cDNA,
A method of connecting this to an appropriate vector and amplifying it in a host cell, selecting a clone having the target red sea bream GH cDNA, and isolating it from the vector possessed by the clone, based on the DNA sequence of the gene of the present invention, For example, the phosphoramidite method (Nature, 310. 105 (198
Examples include methods for chemically synthesizing nucleic acids according to conventional methods such as 4)), and combinations thereof.

The method of preparation and isolation from the pituitary gland of the above-mentioned red sea bream will be described in detail below.

The pituitary gland of red sea bream for obtaining the gene of the present invention can be extracted from either cultured red sea bream or wild red sea bream, but it is preferable to use farmed red sea bream because it is possible to prepare a large number of individuals of uniform size. It's advantageous. The pituitary gland can be extracted by a conventional method, and it is preferable to immediately freeze the pituitary gland using liquid nitrogen, dry ice-ethanol, etc., and then use it for RNA extraction.
Extraction of NA can be carried out using conventional methods, such as the thiocyanine-based anisine-cesium chloride method (Qhirgvin et al.
at.

Biochemistry, idan, 5294 (1
979)), the guanidine thiocyanate-phenol chloroform method, etc. More specifically, in the above guanidine thiocyanate-cesium chloride method, the frozen pituitary gland is first treated with guanidine thiocyanate, sodium citrate, sodium N-lauroylsarcosinate, β-
Mechanically crushed in a mixture of mercaptoethanol, etc.
It is carried out by solubilizing, then layering on an aqueous solution containing cesium chloride, and performing ultracentrifugation. In this way, RNA can be obtained as a precipitate. In the precipitation, rR
Since it contains impurities such as NA% tRNA, RNA having the desired polyA chain is purified according to a conventional method. This can be advantageously carried out using, for example, an oligo dT-cellulose [7 affinity column such as Collaboration Tip Research Co., Ltd.].

The thus obtained poly(A) RNA is used as a template to convert double-stranded DNA (cDNA) using, for example, reverse transcriptase.
It can be used for the synthesis of A).

This CDNA can be synthesized using conventional methods, such as the S1 nuclease method and the Okayama Berg method.
al,,Mo1. Ce1l 3io1. , 3.280
-289 (1983)), but in practical use, for example, CDs commercially available from Amajam Co., Ltd.
This is conveniently carried out using an NA synthesis kit.

Next, in the above method, a DNA sequence encoding the target amino acid sequence of red sea bream GH is isolated from the obtained CDNA. Isolation of this DNA involves inserting the CDNA into an appropriate vector, introducing this vector into a host to amplify it, individualizing it as a plaque or colony of the host containing the DNA sequence, and then selecting the DNA sequence of interest. It is implemented by Examples of vectors used for inserting the CDNA include common ones, such as various plasmids such as various phage DNA1pBR322 derived from λ phage. When the above-mentioned phage DNA is used as a vector, there is an advantage that a large number of candidate strains can be easily prepared and screened. Especially for example λgtl'
0 DNA has the characteristic that only a vector into which a foreign DNA sequence has been inserted forms a plaque against the host cell strain NM514, and can be used more advantageously as the above-mentioned vector.

Insertion of CDNA into the above vector can be carried out by conventional methods such as a method using a synthetic linker or a homopolymer method. For example, after adding EC0RI linkers to both ends of the CDNA, cDNA is cut with the restriction enzyme EC0RI. , this can be inserted into the ECORI cleavage site of the vector, thereby making it possible to obtain the desired recombinant vector. In addition, the resulting recombinant vector can be introduced into a host, and the recombinant vector can be amplified and individualized using various commonly used methods, such as collecting cells mainly in the logarithmic growth phase and treating them with MgSO4. After that, infect cells with crudely modified phage, or infect cells collected in the same way.
This can be carried out by, for example, creating a state in which DNA is naturally easy to incorporate through aCQ2 treatment, and then allowing the vector to be incorporated into this state. Furthermore, the preparation, amplification, and individualization of the above-mentioned recombinant vectors can also be carried out using a commercially available CDNA cloning kit.

Isolation of a recombinant containing a target DNA sequence from a crude recombinant into which a vector containing the CDNA obtained above has been introduced can be carried out by various known methods depending on the type of vector. For example, when using phage DNA as a vector, plaque hybridization
Furthermore, when a plasmid is used as a vector, colony hybridization can be advantageously carried out.

These methods are all described by B.H. Hames and S.J.

Nucleic Acid Hybridization, edited by Higgin S.
id l-5ybridition) J (I
RL Press.

1985>. The second method utilizes the property that recombinant DNA is immobilized on a filter such as a nylon membrane and reacted with a labeled probe, which selectively binds to the target DNA sequence. , to isolate the desired recombinant. Here, the probe is a nucleic acid sequence having a complementary sequence to the target DNA sequence, and may be DNA or RNA, but
It is common and advantageous to use chemically synthesized DNA sequences. The DNA sequence of the probe can be set based on the target partial amino acid sequence of red sea bream GH, and at least a portion of the red sea bream GH gene is considered to have considerable homology with that of the chum salmon GH gene. , known chum salmon G with such high homology)
It is also possible to use the cDNA sequence portion of -J as the above probe.

The gene of the present invention is contained in the thus isolated crude recombinant.

In addition, in the above-mentioned method, various operations employed, such as chemical synthesis of a portion of DNA, enzymatic treatment for the purpose of cutting, deleting, adding or bonding DNA strands, isolation and purification of DNA,
Copying, selection, etc. can be carried out according to conventional methods. More specifically, the isolation and purification of the DNA described above can be carried out by agarose gel electrophoresis, etc., and partial modification of codons in the nucleic acid sequence can be carried out using 5ite-specific Nutaenesis (5ite-Specific Nutaaenes).
is) (Proc, N atl.

Acad, sci, 8U, 5662-5666 (19
84)) etc.

Furthermore, the DNA sequence of the gene of the present invention obtained above can be determined by the Sanger method (YaniSCh-PerrOn et
al.

Gene, 33, 103 (1985)) and Maxam-
Gilbert method [^, H, Haxam and W, G1
1 bert.

Methods in EnxymoloOY, 65.
499-560 (1980)). Note that when determining the DNA sequence of a recombinant obtained as a phage DNA vector, since the vector has a large molecular weight, the CDNA portion of the recombinant described above must be transferred in advance to an appropriate vector such as pUC19, pBR322, etc. It is preferable to determine the DNA sequence after hatching.

Furthermore, the above DNA sequence can be easily determined using a commercially available sequence kit or the like.

According to the above, various documents [for example, J, E.

[) Arnell et al,,” Mo1ecul
ar Ce1l 3io1ogy”5cientif
ic American 3ooks (19F35
), the DNA sequence containing the target red sea bream GH gene contained in the recombinant, the DNA sequence of the gene of the present invention therein, and the amino acid sequence of red sea bream GH can be determined.

DN containing the target red sea bream GH gene thus determined
The A sequence and the corresponding amino acid sequence are as shown in the following formula (5). This is precisely the DNA sequence and amino acid sequence of the red sea bream G)l precursor. That is, this D
The NA sequence is a DNA sequence that encodes an amino acid sequence in which a signal peptide amino acid sequence is added to the N-terminus of the amino acid sequence of red sea bream GH. Red sea bream G)-1 is first biosynthesized as the above precursor, and then the signal peptide is added. It has been revealed that this is a secreted protein from which the peptide portion is removed. The amino acid sequence of the signal peptide portion in the red sea bream GH# precursor amino acid sequence is, for example, Von
He1jne's method (Nucl, Ac1dsRes,
, 14.4683 (1986)).

^TCAGATCCA As a specific example of the vector containing the 7-die G)l gene of the present invention obtained as above, pT
GHEl 1 can be exemplified. The vector pTGHE11
is a plasmid with a size of 3.6 kilobase pairs (kbp), and carries an ampicillin resistance gene as well as a DNA sequence encoding red sea bream GH shown in formula (1) above.

The present inventors transformed the vector pTGHE11 into Escherichia coli J
M109 strain, and the E. coli was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology, as part of the FERM BP Report No. 1457 (FERM BP
1457).

The gene of the present invention can be extracted from the above vector according to a conventional method using restriction enzymes. Further, by using the gene of the present invention, highly pure red sea bream GH can be easily produced in large quantities according to genetic recombination technology.

The production of red sea bream G)f can basically be carried out according to conventional genetic recombination techniques, except for the use of the gene of the present invention. More specifically, a recombinant DNA that allows the gene of the present invention to be expressed in a host cell may be prepared, introduced into the host cell for transformation, and the transformed strain may be cultured.

Here, a representative example of the host cell is Escherichia coli JM10.
9 strains (Vanish-Perron et aJ,,
Qene.

33.103 (1985)), but various other known E. coli strains can also be used as host cells.

The production of red sea bream GH by host cells can be roughly divided into two types: production in the cytoplasm of the host without a signal peptide at the N-terminus, and secretion production from the host cell using a signal peptide. In either case, the N-terminus of the target polypeptide is a methionine (fMet) derived from the start codon, and the C-terminus is a DN encoding
The A sequence is immediately followed by TAASTA as a stop codon.
G or TGA. Therefore, such a start codon and a stop codon are added to the gene of the present invention.

Examples of amino acid sequences encoded by the gene of the present invention provided with the above-mentioned initiation codon are represented by the following formulas (2) and (3).

)1et-ASp-Arg-Val-Val-LeU-
)1et-Lelj-3er-Val-Leu-3er
-Leu-Gly-Val-3er-3er-Gln-
Pro-I 1e-Thr-Asp-Gly-Gln-
Arg-Leu-Phe-3er-I 1e-Ala-
Val-3er-Arp-Val-Gln-His-L
eu-old s-teu-teu-Ala-Gln-Arg
-Leu-Phe-3er-Asp-Phe-Glu-
3er-3er-Leu-Gln-Thr-Glu-G
lu-Gln-Leu-Lys-Leu-Asn-Ly
s-I 1e-Phe-Pro-Asp-Phe-Cy
s-Asn-3er-Glu-Thr-Gln-Arg
-3er-3er-Val-Leu-Lys-Leu-
Leu-3er-11e-3er-Tyr-Arg-L
eu-Val-Glu-3er-Dingrp-Glu-Ph
e-Pro-3er-Arg-3er-Leu-3er
-Gly-Gly-3er-Ala-Pro-Arg-
Asn-Gln-11e-3er-Pro-Lys-L
eu-3er-Glu-Leu-Lys-Net-GJ
y-11e-)1 is-Leu-Leu-11e-A
rg-Ala-Asn-Glu-Asp-Gly-Al
a-Glu-I 1e-Phe-Pro-Asp-3e
r-3er-Ala-Leu-Gln-LetJ-A
a-pro-Dyo-Yr-GIy-ASrl-Dyo-Yr-Dyo-Yr-Gln-3er-Leu-Gly-Ala-As
p-Glu-3er-Leu-Arg-Arp-Dinghr
-■yr-Glu-Leu-Leu-Ala-Cys-
Phe-Lys-Lys-Asp-Het-His-L
ys-Val-Glu-Dinghr-Tyr-Leu-Dingh
r-Val-Ala-Lys-Cys-Arg-Leu
-3er-Pro-Glu-Ala-Asn-cys-
Dinghr-Leu
(2>Net-Gin-Pro-I 1
e-Thr-Asp-Gly-Gln-Arg-Leu
-Phe-3er-I 1e-Ala-Val-3er
-Arg-Val-Gln-His-Leu-11is
-Leu-Leu-Ala-Gln-Arg-Leu-
Phe-3er-Asp-Phe-Glu-3er-3
er-Leu-Gln-Thr-Glu-Glu-Gl
n-Leu-Lys-Leu-Asn-Lys-11e
-Phe-Pro-Asp-phe-cys-Asn-
ser-Asp-■yr-11e-I 1e-3er-
Pro-IIe-Asp-Lys-His-Glu-Dingtlr-Gljl-Arp-3er-3er-Val-
Leu-Lys-Leu-Leu-3er-I 1e-
3er-Tyr-Arg-teu-val-GJu-3
er-■rp-Glu-Phe-Pro-3er-Ar
g-3er-Leu-3er-Gly-Gly-3er
-Ala-Pro-Arg-Asn-GIn-11e-
3er-Pro-Lys-Leu-3er-Glu-L
eu-Lys-Met-Gly-11e-old 5-Leu
-Leu-I 1e-Arg-Ala-Asn-Glu
-Asp-Gly-Ala-Glu-I 1e-Phe
-Pro-Asp-3er-3er-Ala-Leu-
Gln-Leu-Ala-Pro-Dyr-c+y-A
sn-Tyr-Tyr-Gln-3er-Leu-Gl
y-Ala-Asp-Glu-3er-LeU-Arp
-Arp-Thr-Dyo-GIU-LeLl-1eU
-Ala-CyS-Phe-Lys-Lys-Asp-
) 1et-old 5-Lys-Va I-Gl u-Th
r-Dingyr-teu-Dinghr-Val-Ala-Lys
-Cys-Arg-Leu-3er-Pro-Glu-
Ala-Asn-Cys-Thr-Leu (
3) Also, the cDNA of red sea bream GH shown in the above formula (5)
A stop codon (TAG) already exists in the A sequence, and this can be used as the gene of the present invention without introducing a new stop codon.

The above initiation codon can be introduced according to a conventional method, for example, the above formula (
4) can be carried out by cutting the 5' end of the gene of the present invention with an appropriate restriction enzyme, and then ligating thereto a DNA sequence having an appropriate initiation codon. More specifically, the introduction of the start codon can be carried out by, for example, cutting the gene of the present invention represented by the above formula (4) with restriction enzyme 3alI, and then ligating thereto a DNA sequence represented by the following formula (6). . The overlined sequence in this formula is the bosome binding site.

fHetGlnProl 1eThrAspGly5°
CTGGAGGAAAA^^TT^TGCAG
CCG8TCACAG8TGG3°CT^GGACCT
To CTTTTTT AT^CGTCGGCTAGGTGT
The construction of the Mag408 expression vector using the CTACC gene of the present invention can be carried out in accordance with the usual methods commonly used in this type of genetic precomposition technology. Some of the various operations and techniques used in this method are as described above, including cleavage treatment with various restriction enzymes, ligation treatment using T4 DNA ligase, etc., isolation such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc. , purification treatment, recovery by phenol extraction method, purification treatment, etc. The ig of the obtained vector can also be analyzed using conventional methods, such as analyzing its DNA sequence using the Maxam-Gilbert method, or confirming gene introduction and its direction using miniprevention or mapping methods (H, C, Birnboim et al.
, , Nucleic Ac1ds Res, , 7.
1513-1523 (1979)) etc.

There are no particular restrictions on the source vector used for constructing the Mag408 expression vector (it may be of various types known in the art, including, for example, various viral vectors including batataeriophage and animal viruses, and various plasmid,
Includes cosmids and the like. Among these, pBR32 in particular
2 or various established plasmid vectors derived therefrom are suitable.

In addition to the gene of the present invention, the expression vector includes various genetic information necessary for its expression, such as a promoter, in order to actually express the target red sea bream GH when introduced into the host III cell. Information for transcription such as transcription termination signal, poly A chain addition signal (when using eukaryotic cells as a host), and information for translation such as bosome binding site (Shine-Dalgarno sequence, SD sequence) are required. is.

Such information is well known depending on the host cell. For example, as a promoter, t for E. coli is well known.
rp promoter, rec promoter, λPL promoter, llp promoter, tac promoter, etc.
5poi promoter, 5PO2 promoter, pen promoter, etc. for Bacillus subtilis, PH05 promoter for yeast and other eukaryotic cells, promoter for PG,
Examples include GAP promoter, AD)l promoter V40-derived promoter, and the like. The genetic information of these etc. can be isolated by selecting a plasmid etc. containing such etc. and using it as an origin vector when constructing the desired MAG408 expression vector, or from a plasmid etc. containing such etc. according to a conventional method. , can be introduced into a desired vector by chemically synthesizing the vector, and then integrating the vector into an appropriate vector.

In this way, the desired Mag408 expression vector can be obtained.

The present invention also provides such Mag408 expression vectors.

The Mag408 expression vector of the present invention has a set of red sea bream GH expression information units each having a promoter and a ribosome binding site linked upstream of the gene of the present invention, and a transcription termination signal linked downstream, as described above. Characterized by

Furthermore, according to the research of the present inventors, the productivity of the desired red sea bream GH may be increased by using a vector containing two or more sets of the above red sea bream Ge1 expression information units in the same vector. discovered. Therefore, the present invention also provides a vector carrying a plurality of such red sea bream G)1 expression information units.

By introducing (transforming) the red sea bream expression vector of the present invention into an appropriate host cell, the desired red sea bream G1-1 producing ability can be imparted to the host cell. The host cells used here are not particularly limited, and include various known ones, such as gram-positive bacteria such as Escherichia coli, gram-positive bacteria such as Bacillus subtilis, proto-cells such as actinomycetes,
It can be any eukaryotic cell such as yeast, animal or plant cells (Especially 88101 strain derived from 12 strains of E. coli (H, W, Boy).
erand D, Roulland-Dussoi
x,,J,MO+.

3io1. , Dan, 459-472 (1969)) and J.
M103 strain [J, Messtng et al., N
UcIeic Acids Res, 9.309 (
1981) ) is preferred.

Methods for introducing the Red Sea Bream Gl (expression vector of the present invention into the above host cells) and transformation using the same can follow various commonly used methods. For example, the host cells are treated at low temperature in an aqueous solution containing calcium chloride. and adding the vector into the solution (E, Lederberg
and 3. Cohen, J.

3acterio1. ．． 119.1072 (1974
) ) etc.

In this way, cells introduced with the vector of the present invention and transformed can be obtained. The present invention also provides transformed cells that possess the Mag408 expression vector and have the ability to produce red sea bream GH.

The above-mentioned transformed cells can produce and accumulate red sea bream GH by culturing them in a normal cell culture medium. Examples of the medium that can be used for culturing the cells include L medium,
Any of various media such as E medium, M9 medium, M63 medium, etc. can be used. Further, the secondary medium can further contain various commonly known carbon sources, nitrogen sources, inorganic salts, vitamins, natural product extracts, physiologically active substances, and the like.

Culture of transformed cells can be carried out by various methods employing conditions such as p+, temperature, aeration, and stirring that are suitable for the growth of the cells. For example, in the case of Escherichia coli, a pH range of about 5 to 8, especially around pH 7, is appropriate, and it is desirable to culture at a temperature of about 20 to 43°C under aeration and agitation conditions, and there are no particular restrictions on the scale of the culture. do not have. By the above culture, usually about 5~
The desired red sea bream G) (accumulates in the culture suspension at 90rf1).

Red sea bream GH thus accumulated can be separated by normal operations. The operation includes, for example, ultrasonic disruption of cells,
Examples include extraction operations using mechanical disruption, freeze-thaw, osmotic shock, etc., and separation operations of culture supernatants.

Furthermore, the red sea bream GH to be separated as described above can be purified by various purification operations utilizing its physical and chemical properties. The purification operations include, for example, precipitation treatment using a common protein precipitant, ultrafiltration treatment,
This can be carried out by appropriately combining gel filtration treatment, adsorption chromatography treatment, ion exchange chromatography treatment, affinity chromatography treatment, high performance liquid chromatography treatment, and the like.

By the above method, the desired red sea bream GH can be easily produced on an industrial scale with high purity and high yield.

The obtained red sea bream GH was subjected to 5O8-polyacrylamide gel electrophoresis (R, F, 5chleif and p
.

C. Wensink, “Practical Me
thods InMo1ecular Biology
”, sprtnger-ver+ag (1981))
It can be detected by staining with Coomassie brilliant blue or the like.

The red sea bream GH thus obtained is very useful as a growth hormone for red sea bream, particularly in the red sea bream aquaculture industry.

! --51 Hereinafter, the production of the gene of the present invention and the use of the gene for the production of red sea bream GH will be described in more detail with reference to Examples.

Example 1 Preparation of poly(A) RNA from my pituitary gland The pituitary gland was extracted from 480 cultured mugs weighing 900 g and snap-frozen, and the pituitary gland was used as a material for preparation.

The total wet weight of this pituitary gland was approximately 500 m0.

In addition, poly(A) RNA can be prepared using the guanidine thiocyanate-cesium chloride method (Chirgvin et al.
, Biochemistry, 18.5294 (
1979)), as follows.

That is, the pituitary gland was treated with 6M thiocyanine guanidine,
5mM sodium citrate, 0.5% sodium N-lauroyl sarcosinate and 0.5% sodium lauroyl sarcosinate. It was solubilized by crushing (10 strokes) with a Teflon homogenizer in a solution 7.5 consisting of IM β-mercaptoethanol.

This solubilized material was passed through an 18G injection needle several times to fragment the DNA, and then injected at 110,000 rpm in a Hitachi RPR20-20-ter.
The tissue was centrifuged for 10 minutes to remove tissue fragments and proteins, and the supernatant was collected.

Approximately 8 m12 of the collected super-soaked rice was transferred to Beckman 5W40Ti
5,7M which had been placed in a polyaromer tube for
Cesium chloride-0,1M EDTA solution 3 solution 3 sum Q
35000 with Beckman 5w40Ti rotor
After ultracentrifugation at rpm for 18 hours, RNA was collected as a precipitate. After completely removing the supernatant using a 7-spirator, the RNA precipitate was diluted with 10 mM Tris salt II (pH 7,5>
and 1mM EDTA (hereinafter abbreviated as rTEJ) dissolved in 0.9rrIQ, 5M NaC;
0.1 mG was added, incubated at 65°C for 5 minutes, and quenched in water. This mixed liquid 11110 was added in advance to 0.
Oligo (d
T) Cellulose column (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd., column volume 0.35 mQ)
NA was eluted with TE. After 5.5 hours of this eluate,
After adding M NaCQo, 61 mQ and heating in a 65 °C water bath for 5 minutes and quenching in water, this mixture 6.1
1- was applied to the oligo(dT) cellulose column again. The adsorbed RNA was eluted with TE and 0.8μQ
Poly(A) RNA was obtained.

Synthesis of the Tatemi N mome coated cDNA was carried out as follows using a CDI', IA synthesis kit (manufactured by Amajam) according to the protocol attached to the kit.

6.8 μQ of an aqueous solution containing 0.8 μQ of poly(A) RNA
5X first strand synthesis buffer included in the above kit 1-4μQ1 sodium birophosphate solution 1μQ125 single Q human placental ribonuclease inhibitor 1μQ1 deoxynucleoside triphosphate mixture (dA
TP, dGTP, dTTP, dCTP each 10.10.
10.5mM)41! , 2ma/mQ oligo (dT
) Primer 1μQ and 18 units/Kai reverse transcriptase 2
．． Add 2μQ each to make the total amount 20μQ, and heat at 42°C.
The mixture was reacted for 40 minutes to synthesize RNA complementary to the RNA.

Next, add the second strand synthesis buffer included in the above kit to 20μQ of the obtained reaction solution. -37,5μQ,
Add 80.8 units of E. coli ribonuclease, 23 units of E. coli DNA polymerase I, and distilled water to bring the total volume to 99μ.
Q, first at 12℃ for 60 minutes, then at 22℃ for 60 minutes.
0 min and finally 10 min at 70°C, respectively.

After cooling with water, 4 units (1 μQ) of T4 DNA polymerase were added and reacted at 37°C for 10 minutes. This reaction causes R
The RNA part of the NA-DNA double strand is replaced with DNA,
A complete double-stranded CDNA was obtained.

Add 0.25M EDTA (pl(8,0
>10 μQ and 10 μQ of 10% 5DS were added to stop the reaction, the reaction was extracted with phenol-chloroform,
60 μq of double-stranded CDNA was recovered by ethanol precipitation.

Preparation of recombinant vector w4 The CDNA synthesized above was transferred to vector λgt10 (
T., V. Huynh et at, “DNAc
+ontng: Practical approach
h tt, QIOVer I1゜49-78. IR
L Press (1984))
The following procedure was carried out according to the protocol attached to the DNA cloning system (manufactured by Amajam).

To 13 μQ of the aqueous solution containing the total amount of the CDNA synthesized above, 4 μQ of the 5
1 μQ (20 units) of μQSECOR methylase was added to bring the total volume to 20 μQ, and the mixture was incubated at 37°C for 60 minutes to detect the EC0 contained in the above CDNA.
After methylating the RI site, EC0RI methylase was inactivated by heating at 70°C for 10 minutes.

To 20 μQ of the obtained reaction solution, add 10 μQ of the above kit.
XT4DNA ligase buffer? -3μQ1 end phosphorylated EC0RI linker-[5°d(pGGAATTC
C)] 2μQ (1μQ), T4 DNA ligase 2μQ
After adding 3 μQ of distilled water to make 30 μQ, the mixture was reacted at 15°C for 16 hours to form an EC0RI linker at the end of the CDNA.
was added and reacted at 70°C for 10 minutes to inactivate T4 DNA ligase.

To 30 μQ of the obtained reaction solution, add 10 μQ of the above kit.
xEcoRI buffer 10μQ1 restriction enzyme EC0RI
Add 2μQ and 58μQ of distilled water to make 100μQ,
By incubating at 7° C. for 5 hours, CDNA having EC0RI sticky ends derived from the EC0RI linker was obtained.

The obtained reaction solution was applied to a column attached to a CDNA cloning system and eluted with TE containing 100mM NaCQ. This removed the low molecular weight 1 DNA derived from the EC0RI linker. EC0R eluted above
CDNA with I sticky ends was recovered by ethanol precipitation.

To 6μg of an aqueous solution containing half of the cDNA recovered above (30no), IOX ligase buffer 1-1μΩ included in the above kit, λgt70ECORI arm 2μM 1μQ
) and 1 μQ (2.5 units) of T4 DNA ligase were added to bring the total amount to 10 μQ, and the mixture was incubated at 12° C. overnight to obtain crude recombinant λQt10DNA containing CDNA.

This reaction product was recovered by ethanol precipitation and TE2.5
After dissolving in μQ, E. coli BH32688 strain lN20
5recΔ″″(λ 1mm434 CItS b2r
ed3 E am4 Sam7 ) / /1 ] (
D frozen taMIWljh H10μQ and large intestine mBHB2
690 strains [N205recA-(λ 1mm434c
l ts b2 red3 Dam15Sam7)/λ
15 μQ of the ultrasonically disrupted extract was added and incubated at 20° C. for 2 hours, followed by in vitro packaging. The reaction mixture was then supplemented with 100mM NaCQ.
, a solution consisting of 50mM tri-0.01% gelatin 0.5
20μQ of chlorine and chloroform were added.

As described above, recombinant λQt10 DNA containing CDNA was encapsulated in λfur2 particles.

The total amount of the recombinant phage obtained above was prepared using the method of Maniatis et al.

Molecular C1oninp, p63. C
o1d Spring Harbor L aborat
(1982)) host cell N
A suspension of 1 mg (109 cells/Peng) of M514 (manufactured by Amajam) was infected, and approximately 1×10S phage plaques obtained were immobilized on a nylon membrane Biodyne A (manufactured by Nippon Ball).

Next, in the CDNA sequence of chum salmon growth hormone, LeL
50 bases of D corresponding to the region of l-Vat175
NA, that is, the following DNA, was synthesized using a DNA synthesizer model 381A (manufactured by Applied Biosystems), and 32
It was labeled with P and used as a probe.

5' GACGG-CAGGTAGGTC-CGA
CC Ding TG Ding GCATGTC (JrCTrGAAGCA
TGCCA 3゜benton (3enton) and Davis ([)avi
s) method [ibid., “Mo1ecular Cl0
As a result of plaque hybridization at 65°C according to
32 plaques were shown to bind to the 32P-labeled probe. Twelve of the plaques were isolated and named λTGH1 to λTGH12.

Determination of the base of my GHC DNA Phage DNA was prepared from the 12 crude phages obtained above by phenol extraction and ethanol precipitation.

Next, each of the obtained DNAs was treated with restriction enzyme E as follows.
CDN cut with C0RI and retained in the DNA
I got Ae. That is, 100 m of phage DNA 5tlQ
M NaCQ, 50mM Tris-HCl (pH 7.5>,
It was dissolved in an aqueous solution containing 10mM MaSOt and 1mM dithiothreitol (DTT) (hereinafter referred to as "high salt concentration buffer"), and EcoRI (manufactured by Takara Shuzo Co., Ltd.) 50
After adding units to make 300 μQ and reacting at 37° C. for 3 hours, the cleaved DNA was recovered by ethanol precipitation and dissolved in 20 μQ of TE.

Separately, plasmid pUC19 (Yanisch-P
Erron et al., Gene, 33.1
03 (1985)) 3 μQ @ EcoR 120 units of high salt concentration buffer containing 100 μQ, reacted at 37°C for 12 hours, and then ethanol precipitated and recovered DNA.
A solution was prepared by dissolving the following in TE20μQ.

2 μQ of EC0RI fragment solution of the above phage DNA, pLI
c19EcoRI fragment solution 1μQ, IOX fragment-ase buffer [660mM Tris-HCl (pH 7.5>, 66mM
11 μQ of an aqueous solution of MQSO and 100 mM DTT, 1 μQ of 10 mM ATP, 4 μQ of distilled water
and T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) 1μQ (35
0 units) were mixed and reacted at 12°C for 16 hours.

Next, using the above reaction solution, E. coli strain JM109 (Ya
niSCh-Perron et al, (3en
e, 33.

103 (1985)) was transformed. This transformation was performed using the method of Lederberg and Cohen (J, 13acteri).
o1. , 1 'l 9.

1072 (1974)) as follows. That is, suspension 20 of JM109 strain treated with CaCQ2
10 μQ of the above reaction solution was added to 0 μQ, cooled on ice for 60 minutes, heated for 90 seconds in a water bath at 42.5°C, and mixed with broth [1% Batato tryptone, 0.5% yeast extract and 0 μQ]. .5% NaCQ aqueous solution] was added and incubated at 37° C. for 30 minutes. Next, 300 μQ of the obtained suspension was spread on 10 selection plates each and cultured at 37° C. overnight. In addition, as the above selection plate, ampicillin 50μg/1110, isopropyl β-D-thiogalactoside 12.5μQ/1Tl12
and 5-bromo-4-chloro-3-indolyl-β-D
- A plate medium (25 mG/plate) prepared by adding 1.5% agar to L-broth containing 40 μQ/mQ of galactoside and solidifying it was used.

A white colony growing on the plate of the above selective medium was isolated, and plasmid DNA was isolated from the colony. The obtained plasmids are named pTGHE1 to pTGH, respectively, with the same numbers as the names of the recombinant phages from which they are derived.
It was named E12.

The 12 types of plasmids obtained above were treated with various restriction enzymes, such as BplII, EcoRI, Hind m, H
l) aI, PStI, PVuII (all manufactured by Takara Shuzo Co., Ltd.), Ba1I (2y Bongene f%j),
CIaI New England Biolabs 1), SS
A restriction enzyme map of the CDNA contained in each plasmid was created by cutting and analyzing the fragments using T-T (manufactured by Bethesda Research Laboratories, Inc.).

In addition, the length of the CDNA was also estimated.

As a result, the cDNA of pTGHell was the longest, about 9
It consists of 20 bases, Ba1I, BOI■, Cla
I, HindI[I, Ha I, PstI, pvu[,
It was confirmed that each of them had one site that was cleaved by each of the 3stI restriction enzymes.

A restriction enzyme map of the cDNA contained in the above pTGHE11 is shown in FIG.

Furthermore, it was confirmed that the CDNAs contained in each plasmid other than 1) TG)-IE11 described above had the same restriction enzyme map as above.

Next, the entire base sequence of the CDNA of DTGHEl 1 was converted into M1.
Sanger method using 3 phages (Yanisch-pe
rron et al, (3ene, 33.1
03 (1985)), M13 Sequencing Kit (
(manufactured by Toyobo Co., Ltd.).

The result is as shown in equation (5) above.

According to the formula (5), at one end of this base sequence, there is a poly(A) chain that is commonly present in normal mRNA of eukaryotes, and upstream of the poly(A) chain, there is a poly(A>addition signal). A known (5°) AATAAA (3') base sequence exists.

The base sequence also includes a translated region encoding a 203 amino acid sequence. The amino acid sequence is represented by the formula (2
), of which -17 (Met) to -
The amino acid sequence up to 1 (Sep) is from Heijn.
e) method (Nucl, Ac1dsRes, 14.4
683 (1986)), it was confirmed to be a signal peptide of a secreted protein. This means that the red sea bream GH obtained by the present invention is human GH, chum salmon GH,
It has been revealed that, like H and others, it is originally synthesized from producing cells as a precursor with a signal peptide at its N-terminus.

Furthermore, we compared the 186 amino acid sequence (amino acid sequence of red sea bream GH) obtained by excluding the signal peptide from the above amino acid sequence with those of known GH of various animals to examine the homology, and as a result, we found that this red sea bream GH has The amino acid sequence is
It was found to have 68% commonality with chum salmon GH and 33% commonality with human GH. Incidentally, 36% homology is observed between chum salmon GH and human GH.

Furthermore, when the number and position of CyS residues, which are generally considered to be important for the expression of GH activity, were compared for each of the above GHs, complete agreement was found among the three.

From the above, it was confirmed that the above amino acid sequence was that of red sea bream GH.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme map of the cDNA contained in the plasmid pTGH611 of the present invention. (that's all)

Claims (1)

[Scope of Claims] [1] A fish growth hormone gene encoding an amino acid sequence containing a fish growth hormone polypeptide represented by the following formula (1). [There is a gene sequence] (1) [2] The gene according to claim 1, which encodes a polypeptide having the amino acid sequence of either of the following formulas (2) and (3). [There is a gene sequence] (2) [There is a gene sequence] (3) [3] As described in claim 1, in which a stop codon of TAA, TAG, or TGA is further added to the 3' end. genes. [4] The gene according to claim 1, which is isolated as cDNA corresponding to poly(A) RNA prepared from tissues or cells capable of producing fish growth hormone. [5] A fish growth hormone expression vector comprising the gene according to claims 1 to 4. [6] A host cell carrying the vector according to claim 4.