JP2602067B2 - Flounder growth hormone, flounder growth hormone structural gene, recombinant plasmid for flounder growth hormone production, flounder pre-growth hormone, flounder pre-growth hormone structural gene, flounder pre-growth structural gene recombinant plasmid, flounder growth hormone, and method for producing flounder pre-growth hormone - Google Patents

Description

The present invention relates to a recombinant flounder growth hormone, a flounder growth hormone structural gene, a recombinant plasmid for flounder growth hormone production, a flounder pre-growth hormone, a flounder pre-growth hormone structural gene, Field of the Invention The present invention relates to a recombinant plasmid of Japanese flounder pre-growth hormone structural gene, Japanese flounder growth hormone, and a method for producing Japanese flounder pre-growth hormone.

[Prior Art] As in mammals and birds, growth hormone having an amino acid sequence structure specific to each species acts on solid growth in fish, and plays an essential role.

Recent studies have shown that differences in growth hormone structure among fish species are significant. For example, the homology between salmon growth hormone and eel growth hormone is 48% (K. YAMAGUCHI et al., Gen. Comp. Endo
crinol. 66 477 (1987)), the homology between salmon growth hormone and yellowtail growth hormone is 68%, and the homology between yellowtail growth hormone and eel growth hormone is 42%. (M.WATA
HIKI et al., Printing Gen.Comp.Endorinol.70 (1988)).

The homology between these fish growth hormones is 98% homology between human and monkey growth hormone (CHLi et al., Ar
ch. Biochem. Biophys. 245 287 (1986)).

From this point of view, the most natural way to promote growth by growth hormone is to administer growth hormones specific to various species to fish.

Actually, when salmon growth hormone is administered to salmonid fish, an effect of remarkably promoting its growth can be obtained, and thus application to salmon and trout aquaculture has been considered (Japanese Patent Application Laid-Open No. 60-2147).
98).

The application of yellowtail growth hormone to hamachi cultivation is also being considered (Japanese Patent Application Nos. 61-174385 and 61-174385).
184083).

Furthermore, flounder cultivation has also become popular in Japan, and the development and mass production of flounder growth hormone, which promotes the growth of cultured flounder, is one of the most important issues at present.

[Problems to be Solved by the Invention] However, the natural flounder growth hormone is contained only in a trace amount in the flounder pituitary gland, and no structural analysis has been performed.

In view of the above problems, the present inventors have reached the present invention for the purpose of obtaining a large amount of flounder growth hormone using a genetic engineering means.

Furthermore, the present invention elucidates the physiological effects by cloning the flounder growth hormone structural gene, determining the nucleotide sequence, decoding the amino acid sequence structure of flounder growth hormone, and producing flounder growth hormone by Escherichia coli or the like. At the same time, it makes it possible to apply its strong growth promoting effect to flounder aquaculture.

More specifically, the object was to first isolate and amplify the flounder growth hormone structural gene, determine its base sequence, and to decode the amino acid sequence structure of the hormone protein.

Next, the purpose of the present invention was to construct an expression vector by incorporating the structural gene of flounder growth hormone into the gene of a microorganism, and to mass-produce flounder growth hormone by the microorganism.

[Means for Solving the Problems] The flounder growth hormone according to the first invention of the present invention is a peptide having the following structure.

Gln-Pro-Ile-Thr-Glu-Asn-Gln-Arg-Leu-Phe-
Ser-Ile-Ala-Val-Gly-Arg-Val-Gln-Tyr-Leu-
His-Leu-Val-Ala-Lys-Lys-Leu-Phe-Ser-Asp-
Phe-Glu-Asn-Ser-Leu-Gln-Leu-Glu-Asp-Gln-
Arg-Leu-Leu-Asn-Lys-Ile-Ala-Ser-Lys-Glu-
Phe-Cys-His-Ser-Asp-Asn-Phe-Leu-Ser-Pro-
Ile-Asp-Lys-His-Glu-Thr-Gln-Gly-Ser-Ser-
Val-Gln-Lys-Leu-Leu-Ser-Val-Ser-Tyr-Arg-
Leu-Ile-Glu-Ser-Trp-Glu-Phe-Phe-Ser-Arg-
Phe-Leu-Val-Ala-Ser-Phe-Ala-Val-Arg-Thr-
Gln-Val-Thr-Ser-Lys-Leu-Ser-Glu-Leu-Lys-
Met-Gly-Leu-Leu-Lys-Leu-Ile-Glu-Ala-Asn-
Gln-Asp-Gly-Ala-Gly-Gly-Phe-Ser-Glu-Ser-
Ser-Val-Leu-Gln-Leu-Thr-Pro-Tyr-Gly-Asn-
Ser-Glu-Leu-Phe-Ala-Cys-Phe-Lys-Lys-Asp-
Met-His-Lys-Val-Glu-Thr-Tyr-Leu-Thr-Val-
Ala-Lys-Cys-Arg-Leu-Phe-Pro-Glu-Ala-Asn-
Cys-Thr-Leu The flounder growth hormone structural gene according to the second invention of the present invention is a flounder-forming hormone structural gene encoding the following amino acid sequence.

The recombinant plasmid according to the third invention of the present invention contains a flounder growth hormone structural gene encoding the amino acid sequence of the second invention.

The flounder growth hormone structural gene according to the fourth invention of the present invention encodes the following amino acid sequence.

The recombinant plasmid according to the fifth invention of the present invention contains a flounder growth hormone structural gene encoding the amino acid sequence of the fourth invention.

The recombinant flounder pre-growth hormone according to the invention of FIG. 6 of the present invention is a polypeptide having at least the following structure.

Met-Asn-Arg-Val-Ile-Leu-Leu-Leu-Ser-Val-
Met-Cys-Val-Gly-Val-Ser-Ser-Gln-Pro-Ile-
Thr-Glu-Asn-Gln-Arg-Leu-Phe-Ser-Ile-Ala-
Val-Gly-Arg-Val-Gln-Tyr-Leu-His-Leu-Val-
Ala-Lys-Lys-Leu-Phe-Ser-Asp-Phe-Glu-Asn-
Ser-Leu-Gln-Leu-Glu-Asp-Gln-Arg-Leu-Leu-
Asn-Lys-Ile-Ala-Ser-Lys-Glu-Phe-Cys-His-
Ser-Asp-Asn-Phe-Leu-Ser-Pro-Ile-Asp-Lys-
His-Glu-Thr-Gln-Gly-Ser-Ser-Val-Gln-Lys-
Leu-Leu-Ser-Val-Ser-Tyr-Arg-Leu-Ile-Glu-
Ser-Trp-Glu-Phe-Phe-Ser-Arg-Phe-Leu-Val-
Ala-Ser-Phe-Ala-Val-Arg-Thr-Gln-Val-Thr-
Ser-Lys-Leu-Ser-Glu-LeuLys--Met-Gly-Leu-
Leu-Lys-Leu-Ile-Glu-Ala-Asn-Gln-Asp-Gly-
Ala-Gly-Gly-Phe-Ser-Glu-Ser-Ser-Val-Leu-
Gln-Leu-Thr-Pro-Tyr-Gly-Asn-Ser-Glu-Leu-
Phe-Ala-Cys-Phe-Lys-Lys-Asp-Met-His-Lys-
Val-Glu-Thr-Tyr-Leu-Thr-Val-Ala-Lys-Cys-
Arg-Leu-Phe-Pro-Glu-Ala-Asn-Cys-Thr-Leu The flounder pre-growth hormone structural gene according to the seventh invention of the present invention encodes the amino acid sequence of the sixth invention.

The recombinant plasmid according to the eighth invention of the present invention contains a flounder pre-growth hormone structural gene encoding the amino acid sequence of the sixth invention.

The method for producing recombinant flounder growth hormone or recombinant flounder pre-growth hormone according to the ninth invention of the present invention comprises the recombinant plasmid described in the third invention or the recombinant plasmid described in the invention of FIG. A bacterium or a cell into which the recombinant plasmid according to the invention of 8 is introduced is cultured in a culture medium,
A recombinant flounder growth hormone or a recombinant flounder pre-growth hormone according to the first or sixth aspect of the present invention, which is accumulated in the culture medium.

The present inventors have developed pre-growth hormone from flounder pituitary gland.
The mRNA was extracted and its complementary DNA was synthesized oxygenically to form a double-stranded DNA, which was then incorporated into an Escherichia coli plasmid to establish a flounder growth hormone structural gene amplification plasmid.
Next, the recombinant plasmid was introduced into Escherichia coli and cultured, and then the base sequence of the structural gene of flounder growth hormone was determined, and the primary structure (amino acid sequence) of the hormone protein was decoded.

Furthermore, the present inventors incorporated the obtained flounder growth hormone structural gene into an expression vector derived from pBR322 plasmid of Escherichia coli to construct a flounder growth hormone-producing Escherichia coli plasmid. Escherichia coli DH1 strain, JM1
03 strains, JM109 strains (C. Yanisch-Perron et al., Gene, 33 , 103-1
19 (1985)) and established as flounder growth hormone-producing Escherichia coli strains, and then culturing these strains in a medium containing β-D-isopropylthiogalactoside (IPTG). 5 to 10% of protein
And succeeded in producing Japanese flounder growth hormone, and completed the present invention.

 Hereinafter, the present invention will be described in more detail.

The sixth to eighth inventions of the present invention relate to established flounder pre-growth hormone, flounder pre-growth hormone structural gene, and flounder pre-growth hormone structural gene recombinant E. coli plasmid derived from flounder pituitary tissue.

 The above three inventions were established as follows.

Flounder pituitary tissue isolated from flounder is disrupted in guanidinium thiocyanate solution and centrifuged to remove RNA.
Fractions were collected, and the obtained RNA fraction was passed through an oligo d (T) column to concentrate poly (A) ⁺ RNA (messenger RNA) having a poly (A) tail.

The concentrated poly (A) ⁺ RNA was transferred to plasmid pSI4001 (K. Izu
i, et al., Nucleic Acids Res. 14 1615 (1986)), 2.5 kb full length with 77 ampicillin resistance gene and 77 d (T) linkages
And a complementary strand DNA of poly (A) ⁺ RNA is synthesized using reverse transcriptase, and 22 d's are added to its 3 'end by deoxynucleotide terminal transferase.
(C) A chain was attached.

The end of the cloning vector which does not contain the complementary strand DNA is removed by treatment with the restriction enzyme Hind III. On the other hand, both ends have a Hind III adhesion end and 22 d (G) linkages, and one lac promoter and SD (Shine Dalgarno)
A linker derived from pSI4001 having a total length of 390 base pairs and having a sequence and a set of translation initiation codons ATG for three frames was used for Hind III.
Bonded to stump. At the same time, the DNA strand consisting of the vector primer containing poly (A) ⁺ RNA and the linker is circularized by 22 d (C) and d (G) linkages located at both ends, followed by RNaseH and DNA polymerase. I converts the poly (A) ⁺ RNA strand into a DNA strand, forms a phosphodiester bond by DNA ligase treatment, and converts the DNA strand containing flounder growth hormone structural gene (complementary strand DNA) into a complete circular double strand. This was completed as a plasmid with a strand DNA structure.

The present inventors introduced the obtained plasmid into Escherichia coli JM109 strain, cultured at 37 ° C., and grown a colony grown on an agar medium containing acipicillin.
Using the Bcl I fragment as a probe, colony hybridization was performed to obtain a plurality of positive colonies.

After culturing plasmid-introduced Escherichia coli from these positive colonies, the plasmid was extracted and purified, and its length, restriction enzyme map and nucleotide sequence were analyzed to establish a flounder pre-growth hormone structural gene Escherichia coli plasmid pfGH1.

The deoxyribonucleotide sequence from nucleotide number 83 to 655 of the entire nucleotide sequence of pfGH1 shown in Table 2 is the nucleotide sequence of the Japanese flounder pre-growth hormone structural gene first revealed by the present invention, and is shown in FIG. This is the first elucidation of the amino acid sequence of Japanese flounder growth hormone and the amino acid sequence of Japanese flounder growth hormone of claim 1 except for the N-terminal signal peptide portion.

The first to fifth aspects of the present invention relate to flounder growth hormone, flounder growth hormone structural gene, and an Escherichia coli recombinant plasmid for producing flounder growth hormone.

 The above five inventions were obtained as follows.

Nucleotide number 1 of plasmid pfCH1 of the invention of FIG.
Cleavage at position 53 with the restriction enzyme Hae II, the nucleotide number
Purify the 547 bp deoxyribonucleotide chain containing the Japanese flounder growth hormone structural gene obtained by cleavage at position 700 with Nsi I, including the SD sequence and the 5 'end of the Japanese flounder growth hormone gene The synthetic DNA strand was ligated, and the ligated DNA strand was incorporated into a pBR322-derived expression vector containing a pKK223-3 (manufactured by Pharmacia) -derived tac promoter, rrnBT1 and T2 terminators, and an ampicillin resistance gene, thereby producing flounder growth hormone-producing E. coli. Recombinant plasmid (pKFGH3), (pKFGH4)
I got

Recombinant plasmid for producing flatfish growth hormone as described above
pKFGH3 has the cyclic double structure shown in FIG.
Has the entire base sequence shown in Table 1. This flatfish growth hormone producing plasmid pKFGH3 was transformed into E. coli strains DH1, JM109 and JM10.
Flounder growth hormone can be produced in the cells by introducing the strain into 3 strains, JE5505 strain (owned by the National Institute of Genetics), and culturing the introduced Escherichia coli.

When the above plasmid pKFGH3 is constructed, it is constructed by deleting 6 base pairs (corresponding to nucleotide numbers 36 to 41 in Table 1) following the translation initiation codon ATG in the base sequence of the synthetic DNA thus bound. Thus, another flounder growth hormone producing Escherichia coli recombinant plasmid pKFGH4 was obtained.
Depending on the culture of E. coli transfected with pKFGH4, pKFG
Flounder growth hormone can be obtained in which two amino acids following the N-terminal are deleted from flounder growth hormone produced by H3.

By changing the nucleotide sequence of the flounder growth hormone structural gene in the flounder growth hormone producing plasmid thus constructed, a recombinant plasmid producing flounder growth hormone having any desired amino acid sequence can be established.

The ninth invention of the present invention relates to the production of recombinant flounder growth hormone and recombinant flounder pre-growth hormone.

For example, by introducing pKFGH3 into Escherichia coli such as the JM109 strain and the DH1 strain and culturing it in a medium containing β-D-isopropylthiogalactoside (IPTG), the recombinant flounder whose entire amino acid sequence is shown in FIG. 2 is obtained. You can get growth. This recombinant flounder growth hormone is located at the N-terminus of the mature flounder growth hormone from the primary structure of flounder pre-growth hormone shown in FIG. 5, ranging from glutamine at amino acid number 18 to leucine at amino acid number 190. It is composed of 174 amino acids to which methionine is bound.

Similarly, plasmid pKFGH4 is added to E. coli such as JM109 strain.
Alternatively, by introducing pfGH1 and culturing in a medium containing IPTG, recombinant flounder growth hormone consisting of 172 amino acids whose entire amino acid sequence is shown in FIG.
A recombinant flounder pre-growth hormone whose entire amino acid sequence is shown in the figure is obtained. Furthermore, these recombinant flounder pre-growth hormones can be purified as flounder growth hormone by removing the N-terminal with aminopeptidase or the like.

Further, based on the amino acid sequence of flounder growth hormone first elucidated by the present invention, various expression vectors containing a deoxyribonucleotide chain encoding the entire or partial amino acid sequence thereof were constructed, and E. coli was used as an appropriate host. After introduction into Bacillus subtilis, photosynthetic bacteria, yeast or animal cells, the cells are cultured to obtain various recombinant flounder growth hormones.

[Action] The flounder growth hormone in the present invention promotes the growth of flounder, thereby promoting the enlargement of cultured flounder and shortening of the culture period.

[Example] An example of the present invention will be described below.

Example 1 Construction and Cloning of Flounder Pre-growth Hormone Structural Gene Amplification Plasmid 458 mg of flounder pituitary gland was crushed with a polytron in 5 ml of a guanidium thiocyanate (manufactured by Wako Pure Chemical Industries, Ltd.) solution and subjected to 12,000 rotations. After centrifugation for 15 minutes, the RNA fraction obtained by centrifugation in cesium chloride at 29,000 rpm for 19 hours was washed by ethanol precipitation.

The washed RMA fraction was treated with 200 mg of oligo d (T) in a 0.1% sodium dodecyl sulfate (Wako Pure Chemical Industries, Ltd.) solution.
By passing through a column (manufactured by Collaborative Research), 75 μg of total poly (A) ⁺ RNA was obtained. Take 5 μg of poly (A) ⁺ RNA, and add 1.5 μg of vector
Primers (K. Shigesada et al., Gene, 53 163 (1987))
And reacted with 10 units of a reverse enzyme (manufactured by Life Science) at 37 ° C. for 40 minutes.

Deoxynucleotide terminal transferase was allowed to act on the reaction product to add 22 d (C), followed by Hind III (Nippon Gene) treatment. An aliquot of the treatment solution was taken and the linker (K. Shigesada et al., Gene, 53 163 (198
7)) The reaction product was circularized by adding 12 ng, and reacted with RNaseH, DNA polymerase I, and DNA ligase (Takara Shuzo) to obtain a cyclic double-stranded Escherichia coli recombinant plasmid covalently bonded thereto.

Transform the whole amount into E. coli DH1 strain (D.Hanaha
n, J.Mol.Biol. 166 .577 (1983 )) to obtain about 10,000 plasmids introduced colonies. Of the 4000 colonies, 406 base pairs of yellowtail growth hormone cDNA (Japanese Patent Application No. Sho 62)
Colony hybridization was carried out using a Bcl I fragment (T ^* GATCA) of -184083) as a probe to obtain 12 positive colonies. Plasmids from 11 out of 12 colonies contained full-length flounder pre-growth hormone cDNA with the same length and structure. The whole nucleotide sequence of one of these plasmids, pfGH1, was determined to elucidate the structural genes of flounder pre-growth hormone and flounder growth hormone, and the amino acid sequence thereof was also deciphered.

When culturing Escherichia coli such as DH1 and JM109 strains into which the plasmid pfGH1 has been introduced, two purified plasmids per 200 ml of culture solution were obtained.
In this case, a flounder pre-growth hormone structural gene amplified E. coli recombinant plasmid pfGH1 was established.

Escherichia coli JM109 strain into which the above plasmid was introduced was 50 μg / ml.
Grown in culture containing ampicillin at 37 ° C
LB medium (Bacto-tryptone 10g / l, yeast extract 5
g / l, NaCl 5 g / l, pH 7.2-7.4 adjusted with 5N NaOH), the amount was doubled in 30-60 minutes.

Cell count of about to 6-8 hours at the Puraimido introducing 37 ° C. was added E. coli culture 1ml of this medium 200 ml 4 × 10 ⁸ cells / ml
When reached, add chloramphenicol (manufactured by Wako Pure Chemical Industries, Ltd.) to a concentration of 170 μg / ml.
After 8 hours of shaking culture, the number of cells did not increase, but the number of cells increased from 40 copies / cell to 200 / cell, yielding 150 to 300 μg of plasmid pfGH1 per 200 ml medium.

The flatfish pre-growth hormone structural gene recombinant plasmid pfGH1 of the present invention is a 3,820 base pair full-length circular double-stranded DNA containing a deoxyribonucleotide chain encoding flounder pre-growth hormone, a replication origin of pSI4001, and an ampicillin resistance gene.

Tables 2A to 2G also show the entire nucleotide sequence of the plasmid pfGH1 gene. FIG. 4 is a genetic map of the above-mentioned flatfish pre-growth hormone structural gene recombinant plasmid pfGH1, and FIG. 5 is a complete amino acid sequence diagram of the produced flounder pre-growth hormone. It is.

Flounder pre-growth hormone shown in FIG.
It can be administered closer to the form of flounder growth hormone.

Escherichia coli (pfGH1 / JM109) in which the present plasmid pfGH1 was introduced into the JM109 strain as a transformant has been deposited with the National Institute of Microbial Industry and Technology at the National Institute of Advanced Industrial Science and Technology under the deposit number No. 10123 of Microtechnical Laboratory Bacteria.

(Example 2) Construction of recombinant plasmid of flounder growth hormone-producing Escherichia coli The pfGH1 plasmid having the entire nucleotide sequence shown in FIG.
μg, add 20 units of restriction enzyme Hae II, treat at 37 ° C. for 2 hours, add 20 units of restriction enzyme Nsi I
The treatment was performed at 2 ° C. for 2 hours. The reaction DNA sample was electrophoresed in a 5% polyacrylamide slab gel at 150 V for 3 hours to obtain 0.5 μg of a DNA fragment ranging from nucleotide numbers 153 to 700 and having both end structures as described below.

Next, two 54-base DNA strands of the following structure containing two sets of a Shine-Dalgarno sequence (SD sequence) and a cistron starting from the start codon ATG are synthesized using an Applied Biosystems DNA synthesizer 381A. After making it double-stranded, 30 ng of it was taken.

Further, as a vector for the invention, it is derived from the pBR322 plasmid and has rrnBT1 and T2 terminators which are ribosomal RNA transcription termination signals at the 5 ′ end, an ampicillin resistance gene and a replication origin internally, and a tag at the 3 ′ end.
Adjust the double-stranded DNA with the c promoter, and use the restriction enzyme Ps
One unit each of tI and EcoR1 was added, and the mixture was heated at 37 ° C. for 2 hours to make the 5 ′ end a Pst I end and the 3 ′ end to an Est.
coR I stumped. The prepared DNA was subjected to electrophoresis in a 0.8% agarose gel to extract and purify a fragment having a length of 4585 base pairs, and 0.5 µg thereof was taken.

The above three types of DNA fragments were mixed in a solution (20 μ) containing 300 units of T4 DNA ligase, and reacted at 12 ° C. for 16 hours to obtain a circular double-stranded DNA. Take 5μ of this reaction solution, 200
The transformant was introduced into 2 × 10 ⁸ competent E. coli JM109 strains in μ. After 800 μl of LB medium was added thereto and heated at 37 ° C. for 1 hour, 150 μl of this was taken out and spread on a petri dish of LB-agar medium containing 50 μg / ml ampicillin to culture plasmid-introduced Escherichia coli. The emerged colonies are transferred to Whatman 541 filter paper, and a separately prepared Hae II / Nsi I fragment (547 base pairs) of flounder growth hormone cDNA is subjected to a random primer method using [α-32P] CTP (using a kit from Nippon Gene). Labeled DNA
(5 × 10 ⁶ cpm) as a probe in 50% formamide
Colony hybridization was performed by heating to 42 ° C. The paper was treated with 4 × SSC buffer (NaCl 35 g / l, sodium citrate 17.6 g / l) and 2 × SSC buffer (NaCl 17.5 g / l).
g / l, sodium citrate 8.8g / l)
It was exposed to an X-ray film (manufactured by Fujifilm) for 2 days to obtain 22 positive colonies.

Plasmids were extracted from each of the 22 positive colonies and restriction enzyme digests were analyzed. As a result, plasmids from 20 colonies had the same structure. An Escherichia coli strain from one of the colonies was cultured in the presence of β-D-isopropylthiogalactoside (IPTG) (manufactured by Wako Pure Chemical Industries, Ltd.). As described in detail in Example 3, methionine-glutamine type flounder growth hormone A was used. Was produced in more than 5% of the total cell protein. The plasmid pKFGH3 introduced into Escherichia coli is a flounder growth hormone producing Escherichia coli recombinant plasmid having a total length of 5168 base pairs.

Tables 1A to 1 show the entire nucleotide sequence of the gene of the plasmid pKFGH3. FIG. 1 is a structural map of flounder growth hormone producing Escherichia coli plasmid pKFGH3, and FIG. 2 is a complete nucleotide sequence diagram of flounder growth hormone A produced by the plasmid pKFGH3.

In addition, another positive colony had another flounder growth lacking 6 base pairs (corresponding to nucleotide numbers 36 to 41 in Table 1) following the translation initiation codon ATG in the base sequence of the bound synthetic DNA. The hormone-producing Escherichia coli recombinant plasmid pKFGH4 was obtained. By culturing Escherichia coli into which pKFGH4 had been introduced, methionine-isoleucine-type flounder growth hormone B in which two amino acids following the N-terminal were removed from flounder growth hormone A produced by pKFGH3 was obtained. FIG. 3 is an amino acid sequence diagram of recombinant flounder growth hormone B produced from plasmid pKFGH4.

Escherichia coli pKFGH3 / JM109 into which plasmid pKFGH3 was introduced as a transformant was deposited under the accession number No. 10121
E. coli pKFGH4 / JM109 into which plasmid pKFGH4 was introduced
Has been deposited with the Institute of Microorganisms and Industrial Technology of the National Institute of Advanced Industrial Science and Technology under the deposit number No. 10122.

Example 3 Production of Recombinant Flounder Growth Hormone First, the JM109 E. coli strain introduced with the flounder growth hormone-producing recombinant plasmid pKFGH3 shown in Example 2 was transformed into 5 strains.
M-9 containing 0 μg / ml ampicillin and 0.2% casamino acid
5 ml of the medium was inoculated and cultured at 37 ° C. The turbidity of the culture is 0D6
When 00 = 0.2, it is an inducer of the lac promoter. β-D-isopropylthiogalactoside (IPTG)
2 mM was added, and the culture was continued at 37 ° C. for 5 hours. The obtained culture was centrifuged at 12000 rpm for 10 minutes to collect the cells. The cells were used as Laemmli sample lysates (12.5 mM Tris-HCl, 0.02% BPB, 15% glycero).
1, 350 mM β-ME, pH = 6.8) and heated at 100 ° C. for 10 minutes, followed by the method of Remmli (UKLaemmli, Nature, 227).
SDS-polyacrylamide gel electrophoresis according to 680 (1980)) and the electrophoresed gel was run in acetic acid / methanol in Coomassie Brilliant Bl.
ue) to confirm the production of flounder growth hormone.

The flounder growth hormone A protein produced in this way reached a concentration of 5% or more of the total bacterial cell protein as measured by a densitometer (manufactured by Shimadzu Corporation).

Next, the Escherichia coli producing the flounder growth hormone A was subjected to SDS-polyacrylamide gel electrophoresis, and the electrophoresed gel was subjected to a Burnett method (WNBurnette, Anal. Bioche.
m. 112 195 (1981)). Then, the nitrocellulose membrane was treated by an enzyme immunoassay using an antibody against flounder growth hormone, a protein A / horseradish peroxidase binding reagent and 4-chloro-1-naphthol, and flounder growth hormone A was placed at the same position with a molecular weight of about 20,000. Band could be detected.

Further, a protein having a molecular weight of about 20,000 separated by SDS-polyacrylamide gel electrophoresis was extracted from the gel, and the N-terminal amino acid sequence was analyzed using a gas-phase protein sequencer. The sequence was obtained. As a result, plasmid pKFG
It was confirmed that Escherichia coli carrying H3 produced flounder growth hormone A.

In a similar manner, Escherichia coli transfected with plasmid pKFGH4 or Escherichia coli transfected with plasmid pfGH1 can be cultured to produce flounder growth hormone or flounder pre-growth hormone.

[Effects of the Invention] As described above, the present invention is based on the amino acid sequence of Japanese flounder growth hormone firstly elucidated by the present invention, and includes various types including a deoxyribonucleotide chain encoding the entire or partial amino acid sequence thereof. Expression vectors are constructed, and each is transformed into an appropriate transformant into Escherichia coli, Bacillus subtilis, photosynthetic bacteria, yeast, animal cells, etc., and then cultured to facilitate the production of recombinant flounder growth hormone from various transformants. Moreover, it can be obtained in large quantities. Further, as a specific example, plasmid pKFGH3, plasmid pKFGH4, and plasmid pfGH1 are cultured to obtain flounder growth hormone and flounder pre-growth hormone, and to promote the growth of flounder, thereby promoting the enlargement of cultured flounder and shortening of the culture period. There is an effect that can be done.

[Brief description of the drawings]

FIG. 1 is a structural map of a flounder growth hormone producing Escherichia coli recombinant plasmid pKFGH3, FIG. 2 is a complete amino acid sequence diagram of flounder growth hormone A produced by the plasmid pKFGH3, FIG.
The figure shows flounder growth hormone producing E. coli recombinant plasmid pK.
Fig. 4 is an amino acid sequence diagram of recombinant flounder growth hormone B produced from FGH4, Fig. 4 is a genetic map of a flounder pre-growth hormone structural gene recombinant plasmid pfGH1, and Fig. 5 is a complete amino acid sequence diagram of produced flounder pre-growth hormone. is there.

 ──────────────────────────────────────────────────続 き Continued on the front page (54) [Title of the invention] Japanese flounder growth hormone, Japanese flounder growth hormone structural gene, recombinant plasmid for Japanese flounder growth hormone production, Japanese flounder pre-growth hormone, Japanese flounder pre-growth hormone structural gene, Japanese flounder pre-growth structural gene Method for producing recombinant plasmid, flounder growth hormone and flounder pre-growth hormone

Claims (9)

(57) [Claims] 1. A flounder growth hormone which is a polypeptide having the following structure. Gln-Pro-Ile-Thr-Glu-Asn-Gln-Arg-Leu-Phe-
Ser-Ile-Ala-Val-Gly-Arg-Val-Gln-Tyr-Leu-
His-Leu-Val-Ala-Lys-Lys-Leu-Phe-Ser-Asp-
Phe-Glu-Asn-Ser-Leu-Gln-Leu-Glu-Asp-Gln-
Arg-Leu-Leu-Asn-Lys-Ile-Ala-Ser-Lys-Glu-
Phe-Cys-His-Ser-Asp-Asn-Phe-Leu-Ser-Pro-
Ile-Asp-Lys-His-Glu-Thr-Gln-Gly-Ser-Ser-
Val-Gln-Lys-Leu-Leu-Ser-Val-Ser-Tyr-Arg-
Leu-Ile-Glu-Ser-Typ-Glu-Phe-Phe-Ser-Arg-
Phe-Leu-Val-Ala-Ser-Phe-Ala-Val-Arg-Thr-
Gln-Val-Thr-Ser-Lys-Leu-Ser-Glu-Leu-Lys-
Met-Gly-Leu-Leu-Lys-Leu-Ile-Glu-Ala-Asn-
Gln-Asp-Gly-Ala-Gly-Gly-Phe-Ser-Glu-Ser-
Ser-Val-Leu-Gln-Leu-Thr-Pro-Tyr-Gly-Asn-
Ser-Glu-Leu-Phe-Ala-Cys-Phe-Lys-Lys-Asp-
Met-His-Lys-Val-Glu-Thr-Tyr-Leu-Thr-Val-
Ala-Lys-Cys-Arg-Leu-Phe-Pro-Glu-Ala-Asn-
Cys-Thr-Leu 2. A flounder growth hormone structural gene encoding the following amino acid sequence. 3. A recombinant plasmid containing a flounder growth hormone structural gene encoding the amino acid sequence of claim 2. 4. A flounder growth hormone structural gene encoding the following amino acid sequence. 5. A recombinant plasmid containing a flounder growth hormone structural gene encoding the amino acid sequence of claim 4. 6. A recombinant flounder pre-growth hormone which is a polypeptide having at least the following structure. Met-Asn-Arg-Val-Ile-Leu-Leu-Leu-Ser-Val-
Met-Cys-Val-Gly-Val-Ser-Ser-Gln-Pro-Ile-
Thr-Glu-Asn-Gln-Arg-Leu-Phe-Ser-Ile-Ala-
Val-Gly-Arg-Val-Gln-Tyr-Leu-His-Leu-Val-
Ala-Lys-Lys-Leu-Phe-Ser-Asp-Phe-Glu-Asn-
Ser-Leu-Gln-Leu-Glu-Asp-Gln-Arg-Leu-Leu-
Asn-Lys-Ile-Ala-Ser-Lys-Glu-Phe-Cys-His-
Ser-Asp-Asn-Phe-Leu-Ser-Pro-Ile-Asp-Lys-
His-Glu-Thr-Gln-Gly-Ser-Ser-Val-Gln-Lys-
Leu-Leu-Ser-Val-Ser-Tyr-Arg-Leu-Ile-Glu-
Ser-Typ-Glu-Phe-Phe-Ser-Arg-Phe-Leu-Val-
Ala-Ser-Phe-Ala-Val-Arg-Thr-Gln-Val-Thr-
Ser-Lys-Leu-Ser-Glu-Leu-Lys-Met-Gly-Leu-
Leu-Lys-Leu-Ile-Glu-Ala-Asn-Gln-Asp-Gly-
Ala-Gly-Gly-Phe-Ser-Glu-Ser-Ser-Val-Leu-
Gln-Leu-Thr-Pro-Tyr-Gly-Asn-Ser-Glu-Leu-
Phe-Ala-Cys-Phe-Lys-Lys-Asp-Met-His-Lys-
Val-Glu-Thr-Tyr-Leu-Thr-Vol-Ala-Lys-Cys-
Arg-Leu-Phe-Pro-Glu-Ala-Asn-Cys-Thr-Leu [7] A flounder pre-growth hormone structural gene encoding the amino acid sequence of [6]. 8. A recombinant plasmid containing a flounder pre-growth hormone structural gene encoding the amino acid sequence of claim 6. 9. A bacterium or a cell into which the recombinant plasmid according to claim 3 or the recombinant plasmid according to claim 5 or the recombinant plasmid according to claim 8 has been introduced, is cultured in a culture medium. A method for producing a recombinant flounder growth hormone or a recombinant flounder pre-growth hormone, comprising separating and purifying the recombinant flounder growth hornmon or the recombinant flounder pre-growth hormone according to claim 1 or 6.