JPH057995B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a DNA encoding a fish growth hormone polypeptide, and a recombinant product incorporating the DNA.
The present invention relates to DNA, a microorganism containing the recombinant DNA, and a method for producing a fish growth hormone polypeptide using the microorganism. Fish growth hormones are expected to have wide applications in the fish aquaculture industry. BACKGROUND OF THE INVENTION Mammalian growth hormones are produced in the pituitary gland, and their activity and structure are known. For example, for human growth hormone,
J.Am.Chem.Soc., 80 , by UJLewis et al.
4429 (1958) by AShartree in Biochem.
J., 100 , 754 (1966), by CHLi et al. in Arch.
Biochem. Biophys. Acta (Suppl.), 1 , 327 (1962)
has been reported. There have been many reports that fish growth hormones have been isolated, but there are only a few that are reliable in terms of their physiological activity and protein chemical properties. Examples of reliable reporting include: Isolation example from tilapia SWFarmer et al., Gen.
Comp. Endocrin., 30 , 91 (1976). Isolation example from a sturgeon shark SW Farmer et al., Endocrinology, 108 , 377 (1981). Isolation example from carp AFCook et al., Gen. Comp. Endorcrin., 50 , 335 (1983). On the other hand, regarding mammalian growth hormone genes, there are the rat growth hormone gene [PHSeeburg et al.: Nature 270 486 (1977)], the bovine and pig growth hormone genes [PHSeeburg et al.: DNA, 2 ,
37 (1983)], human growth hormone gene [JA
Martial et al.: Sciece, 205 , 602 (1979)], but there are no reports yet on growth hormone genes in fish. The present inventors previously extracted and purified growth hormone from salmon pituitary gland, and determined the amino acid sequence (31) from the N-terminus. It has also been confirmed that this substance has a growth-promoting effect on teleost fish [Patent Application 1986-68670]. Problems to be Solved by the Invention Fish growth hormone has a growth-promoting effect on fish, and is therefore useful as a composition for fish feed, but the supply of it from the pituitary gland of fish is limited. . Therefore, it is desired to develop a method for supplying fish growth hormone in large quantities at low cost. Means for Solving the Problems The present inventors conducted research on a method for producing fish growth hormone using recombinant DNA technology. As a result, we succeeded in collecting DNA complementary to fish growth hormone polypeptide, which can be used in the production of fish growth hormone, and in producing recombinant DNA and microorganisms containing this DNA. That is, Metsenjar RNA from the salmon pituitary gland.
(mRNA) and complementary DNA to this
Cloning the salmon growth hormone gene by synthesizing (cDNA), then synthesizing a DNA probe corresponding to the amino acid sequence near the N-terminus of salmon growth hormone, and selecting cDNA that hybridizes with this DNA. succeeded in. Furthermore, the entire base sequence of this cDNA was determined, leading to the completion of the present invention. The present invention will be explained in detail below. The present invention provides fish growth hormone polypeptides,
In particular, polypeptides having the peptide sequences shown in Table 1 are provided. The polypeptide can be produced using recombinant DNA techniques as described below. That is, DNA (cDNA) complementary to fish growth hormone mRNA is used as a template.
A recombinant plasmid incorporating the cDNA is prepared. Furthermore, the recombinant plasmid is inserted into a host microorganism. The DNA and recombinant plasmid can be used to amplify the salmon growth hormone gene, especially in bacteria such as E. coli. Microorganisms having the recombinant plasmid are useful for producing salmon growth hormone in large quantities at low cost. Accordingly, the present invention provides a DNA encoding a fish growth hormone polypeptide, a recombinant DNA incorporating the DNA, and a microorganism containing the recombinant DNA. The DNA and recombinant plasmid of the present invention are prepared by the following general method. Total RNA was prepared from the pituitary gland of chum salmon, and this was added to oligo dT cellulose.
RNA containing polyadenylic acid (polyA) (polyA ⁺ RNA) is separated by passing it through a column.
Using this polyA ⁺ RNA as a template, double-stranded DNA is synthesized using reverse transcriptase. Recombinant in vitro
E. coli plasmids using recombinant DNA techniques
It can be obtained by inserting the synthetic DNA into a vector DNA such as DNA. Chum salmon growth hormone mRNA
Select a recombinant plasmid that has DNA complementary to . Next, the method for producing the DNA and recombinant plasmid of the present invention will be specifically explained. The pituitary gland is removed from a captured chum salmon and immediately frozen in liquid nitrogen. Guanidium isothiocyanate is added to the frozen pituitary gland to crush it and solubilize it. Next, it is layered on a CsCl solution layer, and after ultracentrifugation, it is precipitated and total cytoplasmic RNA is obtained. Alternatively, only RNA can be precipitated and recovered by adding LiCl to the guanidium isothiocyanate solubilized product. Dissolve the extracted RNA in a high salt concentration (e.g. 0.5M) solution of NaCl or KCl and add oligo(dT)
The mRNA containing poly(A) is adsorbed onto the column by passing it through a cellulose column. The mRNA with poly(A) is isolated by elution using a low salt solution such as water, 10mM Tris-HCl buffer. The following is the Okayama-Berg method [Okayama
&Berg; Mol. Cell. Biol. 2 , 161 (1982)], synthesis of cDNA and its integration into a vector are performed. First, vector primers are synthesized. As a vector, for example, pCDV1 can be dissolved in a suitable solution, such as Tris-HCl buffer (e.g. PH7.5,
10mM), _MgCl2 (e.g. 6mM), and NaCl (e.g. 10mM),
Cut pCDV1 at the KpnI site. This DNA was added to Tris-HCl buffer (e.g. PH6.8, 30mM), sodium cacodylate (e.g. 140mM), CoCl ₂
(e.g. 1mM), dithiothreitol (e.g. 0.1mM) and dTTP (e.g. 0.25mM).
Approximately 60 thymidyl residues are added to both 3' ends of the vector DNA by incubation with terminal deoxynucleotidyl transferase at a constant temperature (eg, 37°C) for a certain period of time (eg, 20 minutes). Furthermore, this DNA was cut with EcoRI in a solution containing Tris-HCl buffer (e.g. PH7.5, 10mM), MgCl ₂ (e.g. 6mM), NaCl (e.g. 100mM), and then subjected to low melting point agarose gel electrophoresis [Lars Wieslander: Analytical
Biochemistry, 98 , 305 (1979)], and approximately
Recover a 3.1 kilobase fragment. Then the DNA
High salt concentration of NaCl or KCl (e.g. 0.5M)
It is dissolved in a solution and passed through a poly(dA) cellulose column, so that only vector primer molecules having poly(T) are adsorbed onto the column. Elution is performed using a low salt solution such as water, 10mM Tris-HCl buffer to isolate only the poly(T)-attached vector primer molecules. Next, linker DNA is synthesized. for example
pL1DNA in a suitable solution, e.g. Tris-HCl.
The PstI site of pL1 is cleaved by treatment with PstI in a solution containing a buffer (eg, PH7.5, 10mM), MgCl ₂ (eg, 6mM), and NaCl (eg, 50mM).
This DNA is treated in the same manner as for vector primer synthesis, except that dGTP is added instead of dTTP, and approximately 15 oligo dG chains are added. Applicable
Transfer the DNA to a suitable solution such as Tris-HCl buffer (e.g. PH7.5, 10mM), _MgCl2 (e.g.
6mM), in a solution containing NaCl (e.g. 60mM)
Cut at Hind. Fractionate approximately 0.5 kilobase DNA fragments by agarose gel electrophoresis.
Collect with DEAE paper. In this way, linker DNA is obtained. Using poly(A) ⁺ RNA, vector primer, and linker DNA obtained as above, cDNA
Perform synthesis. Poly(A) ⁺ RNA, vector primer DNA was dissolved in Tris-HCl buffer (e.g. PH
8.3, 50mM), _MgCl2 (e.g. 8mM), KCl (e.g. 30mM), dithiothreitol (e.g.
0.3mM), dATP, dTTP, dCTP, dGTP (e.g. 2mM each), reverse transcriptase was incubated at a constant temperature (e.g. 37°C) for a certain period of time (e.g. 40°C).
(minutes). Approximately 15 oligo dC strands are added to the 3' end of the RNA-DNA duplex thus obtained using the same procedure as when adding dT strands to the vector primer, except that dTTP is changed to dCTP.
This DNA was added to a Tris-HCl buffer (e.g. PH
7.5, 10mM), _MgCl2 (e.g. 6mM), NaCl (e.g. 60mM).
Mix the previously prepared linker DNA with this DNA and add Tris-HCl buffer (e.g. PH7.5,
20mM), _MgCl2 ( _e.g. 4mM), ( _NH4 ) _2SO4
(e.g. 10mM), KCl (e.g. 0.1M), β-nicotinamide adenine dinucleotide (β-
NAD) (e.g. 0.1mM) with E. coli DNA ligase for a fixed period of time (e.g. 16
time) and incubate at a constant temperature (e.g. 12°C). In this way, cDNA and linker DNA are circularized. This reaction solution contains dATP,
Add dTTP, dGTP, and dCTP to a final concentration of 40 μM each, and add E. coli DNA ligase and E. coli DNA.
A recombinant plasmid containing a complete double-stranded cDNA is obtained by adding a polymerase, E. coli ribonuclease H, and converting the RNA portion into DNA. Using the thus obtained recombinant plasmid, E. coli
For example, Escherichia coli c600SF8 strain is transformed by the method of Scott et al. [Katsuya Shigesada: Cell Engineering 2 , 616 (1983)]. Since the ampicillin resistance gene is present on the recombinant plasmid obtained above, the transformed E. coli exhibits ampicillin resistance. The following method uses a novel recombinant plasmid containing a gene complementary to fish growth hormone mRNA from these ampicillin-resistant ( ^Apr ) strains.
Commonly used to select strains that carry DNA. That is, the transformed strain obtained above is immobilized on a nitrocellulose filter and allowed to associate with a synthetic DNA probe having a DNA sequence predicted from the known amino acid sequence of chum salmon growth hormone, and those with strong association are selected. Grunstein-Hogness method, Proc. Natl. Acap.
Sci., USA., 72 , 3961 (1975)]. probe dna
is the usual triester method (J.Am.Chem.Soc.,
97, 7327 (1975)]. Selection using synthetic DNA probes was performed using the method of Southern et al. [J. Mol.
Biol. 98 , 503 (1975)], and by this method a recombinant plasmid containing a gene complementary to chum salmon growth hormone mRNA was obtained.
DNA can be identified. The novel recombinant plasmids of the present invention can be used for large scale production of fish growth hormone polypeptides by microorganisms such as E. coli or eukaryotic cells. Examples of the present invention are shown below. Example 1 Poly A ⁺ RNA from chum salmon pituitary gland
Preparation: Guanidium thiocyanate-cesium chloride method from chum salmon pituitary gland [Maniatis et al., eds.
Molecular Cloning.p196，Cold Spring Harbor
PolyA-containing RNA was prepared as follows . 2g frozen pituitary gland of chum salmon (about 30 individuals)
4M guanidinium thiocyanate, 0.5% sarcosine, 5mM sodium citrate (PH7) and
10ml of a solution consisting of 0.1M β-mercaptoethanol
The mixture was crushed and solubilized using a Teflon homogenizer (5 rpm). This homogenate was passed through an 18G needle several times to fragment the DNA. 5.7MCsCl, 0.1M
1.2 ml of each EDTA (PH8) solution was dispensed into ultracentrifuge tubes, and the homogenate was layered thereon.
After centrifugation at 35,000 rpm for 15 hours in a Hitachi RPS40 rotor, RNA was collected as a precipitate. The RNA precipitate was dissolved in 10 ml of Tris-HCl (PH8.0) solution containing 1 mM EDTA, extracted with phenol-chloroform, and recovered by ethanol precipitation. obtained
Approximately 1 mg of RNA was added to 10 mM Tris-HCl (PH8.0) and
It was dissolved in 1 ml of a solution consisting of 1 mM EDTA. 65
℃, incubate for 5 minutes and add 0.1 ml of 5M NaCl.
added. The mixture was subjected to chromatography on an oligo dT cellulose column (manufactured by PL Biochemicals). with adsorbed polyA
mRNA was purified using 10mM Tris-HCl (PH8.0 and 1mM
Contains polyA eluted with a solution consisting of EDTA
Approximately 10 μg of mRNA was obtained. Example 2 cDNA synthesis and insertion of the DNA into a vector: Okayama-Berg method [Mol.Cell.Biol., 2 ,
161 (1982)], cDNA was synthesized and a recombinant plasmid incorporating it was constructed. An outline of the process is shown in FIG. pCDV1〔Okayama & Berg: J.Mol.Cell.
Biol., 3 , 280 (1983)] 400μg in 10mM Tris-
Add to 300 μl of a solution consisting of HCl (PH7.5), 6 mM MgCl ₂ and 10 mM NaCl, plus 500 units of Kpn.
(manufactured by Takara Shuzo Co., Ltd.) and reacted at 37℃ for 6 hours.
It was cut at the Kpn site in the plasmid. After phenol-chloroform extraction, ethanol precipitation
DNA was recovered. About the Kpn-cleaved DNA
200μg was added to a buffer solution (hereinafter abbreviated as TdT buffer) consisting of 40mM sodium cacodylate, 30mM Tris-HCl (PH6.8), 1mM CaCl ₂ and 0.1mM dithiothreitol (hereinafter abbreviated as DTT).
Add 200 μl of a solution containing dTTP to 0.25 mM, and further add 81 units of terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT) (manufactured by PL Bioche-micals).
The reaction was carried out at 37°C for 11 minutes. Here, Kpn of pCDV1
Approximately 67 poly-dT chains were added to the 3' end of the cleavage site. Approximately 100 μg of pCDV1 DNA with poly dT chain added was recovered from the solution by phenol-chloroform extraction and ethanol precipitation. the DNA
10mM Tris-HCl (PH7.5), 6mM _MgCl2 ,
In addition to 150 μl of a buffer consisting of 100 mM NaCl, 360 units of EcoR (manufactured by Takara Shuzo Co., Ltd.) was added and incubated at 37°C.
The reaction was allowed to proceed for 2 hours. After the reaction product was subjected to low melting point agarose gel electrophoresis, a DNA fragment of about 3.1 Kb was collected,
Approximately 60 μg of poly-dT chain-added pCDV1 was obtained. the DNA
10mM Tris-HCl (PH8.0) and 1mM
It was dissolved in 500 μl of a solution consisting of EDTA, incubated at 65° C. for 5 minutes, cooled on ice, and 50 μl of 5M NaCl was added. The mixture was subjected to chromatography on an oligo dA cellulose column (manufactured by Collaborative Research). Poly-dT with sufficient chain length is adsorbed onto the column and added to 10mM Tris-HC (PH
8.0) and 1mM EDTA,
27 μg of pCDV1 (hereinafter abbreviated as vector primer) to which a poly-dT chain was added was obtained. Next, prepare linker DNA. pL1 [Okayama & Berg: Mol.Cell.Biol. 3 ,
280 (1983)] Approximately 14 μg was added to 10 mM Tris-HCl (PH
7.5), in addition to 200 μl of a buffer consisting of 6 mM MgCl ₂ and 50 mM NaCl, 50 units of Pst (manufactured by Takara Shuzo Co., Ltd.) was added and reacted at 37°C for 4 hours.
It was cut at the Pst site in pL1DNA. After the reaction product was extracted with phenol-chloroform, ethanol precipitation was performed, and about 13 μg of pL1 DNA cut with Pst was recovered. Approximately 13 μg of the DNA was added to TdT buffer at a final concentration.
Add to 50μl of solution containing 0.25mM dGTP, and
54 units of TdT (manufactured by PL Biochemicals) was added and incubated at 37°C for 13 minutes to add about 14 dG chains to the 3' end of the Pst cleavage site of pL1. After phenol-chloroform extraction, ethanol precipitation
DNA was recovered. The DNA was mixed with 100 μl of 10 mM Tris-HCl (PH7.5), 6 mM MgCl ₂ and 60 mM
In addition to 100 μl of a buffer consisting of NaCl, 80 units of Hind (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was incubated at 37° C. for 3 hours, and the pL1 DNA was cleaved at the Hind site. The reaction product was fractionated by agarose gel electrophoresis, and a DNA fragment of about 0.5 Kb was separated using the DEAE paper method [Dretzen et al., Ana1.Biochem., 112 , 295 (1981)].
Linker DNA with oligo dG strand
(hereinafter simply referred to as linker DNA) was obtained. Approximately 2 μg of poly(A) RNA prepared above and approximately 1.4 μg of vector primer were mixed with 50 mM Tris-HCl (PH
8.3), 8mM _MgCl2 , 30mM KCl, 0.3mM
DTT, 2mM dNTP (dATP, dTTP, dGTP and dCTP) and 10 units of ribonuclease inhibitor (manufactured by PL Biochemicals) were dissolved in 22.3 μl of a solution, and 10 units of reverse transcriptase (manufactured by Seikagaku Corporation) were added. In addition, the mixture was incubated at 37°C for 40 minutes to synthesize DNA complementary to the mRNA. The reaction product was extracted with phenol-chloroform,
Ethanol precipitation was performed to recover vector primer DNA with an RNA-DNA double strand added thereto.
The DNA contained 66 μM dCTP and 0.2 μg polyA.
Dissolve 14 units of TdT (P-L) in 20 μl of TdT buffer.
Biochemicals) and incubated at 37°C for 8 minutes to add 12 dC strands to the 3' end of the cDNA. The reaction product was extracted with phenol-chloroform, and dC chains were added by ethanol precipitation.
cDNA-vector primer DNA was recovered.
The DNA was dissolved in 400 μl of a solution consisting of Tris-HCl (PH7.5), 6 mM MgCl ₂ and 60 mM NaCl, and incubated for 20 min.
A unit of Hind (manufactured by Takara Shuzo Co., Ltd.) was added, incubated at 37°C for 2 hours, and cut at the Hind site.
The reaction product was extracted with phenol-chloroform and precipitated with ethanol to obtain 0.5 pmole of dC strand-added cDNA-vector primer DNA. Applicable
0.08pmole of DNA and the above linker
0.16pmole of DNA in 40μl Tris-HCl (PH7.5),
Solution consisting of 0.1M NaCl and 1mM EDTA
Add to 40 μl and incubate at 65℃, 42℃, and 0℃ for 10 minutes each.
Incubated for 25 and 30 minutes. 20mM Tris-HCl (PH7.5), 4mM _MgCl2 , 10mM
( _NH4 ) _2SO4 , _0.1M KCl and 0.1mM β-NAD
A reaction solution was prepared with the following composition in a total volume of 400 μl. 10 units of Escherichia coli DNA ligase (manufactured by New England Biolabs) was added to the reaction solution, and the solution was incubated at 11°C. The reaction solution was diluted with 40 μM each.
dNTP, 0.15mM β-NAD, 5 units of E. coli DNA ligase, 7 units of E. coli DNA polymerase (P-L).
Biochemicals) and 2 units of Escherichia coli ribonuclease H (P-L Biochemicals) were added and incubated at 12°C and 25°C for 1 hour each. In the above reaction, recombinant DNA containing cDNA
circularization and the RNA part of the RNA-DNA duplex
DNA was replaced, and a complete double-stranded DNA recombinant plasmid was generated. Example 3 Selection of recombinant DNA containing chum salmon growth hormone cDNA: Using the recombinant plasmid obtained in Example 2, Escherichia coli c600SF8 strain [Cameron: Proc. Natl. Acad.
Sci. USA, 72 , 3416 (1975)] was transformed according to the method of Scott et al. [Katsuya Shigesada: Cell Engineering, 2 , 616 (1983)]. Of the approximately 10,000 colonies obtained,
4800 were immobilized on nitrocellulose. Synthetic DNA corresponding to the 23rd to 28th amino acid sequence from the N-terminus of chum salmon growth hormone, i.e. (3rd base is A or G, 9th base is T or C, 12th base is C or T, 15th base is C or T)
Eight bacterial strains were selected that strongly associated with the ³² P-labeled probe at 40°C (combining to form a mixture of 16 synthetic DNAs) [Grunstein-
Hogness' method, Proc.Natl.Acad.Sci.USA, 72 ,
3961 (1975)]. Regarding the eight strains obtained, Southern
By the method of [J.Mol.Biol., 98 , 503 (1975)],
Synthetic DNA probe corresponding to the above probe and the amino acid sequence near the C-terminus (The 3rd base is C or T, the 6th base is A or G, the 9th base is A, T, G, or C, and the 12th base is G or A, which can be combined to create a mixture of 32 synthetic DNAs.) A meeting was confirmed with Naru.
These plasmids are pSGH1, 3, 6, 8, 9,
10, 14, and 17, all of which are DNA predicted from the amino acid sequence of chum salmon growth hormone.
It was thought that it contained the growth hormone cDNA because it had the same sequence. Example 4 Base sequence of the plasmid pSGH1: The eight plasmids obtained above were digested with various restriction enzymes, and the cleavage map of the cDNA portion was determined. Based on the location of the restriction enzyme site, the obtained plasmids can be classified into three groups: pSGH1, 6, 9,
They were divided into groups 10 and 17, pSGH3 group, and pSGH8 and 14 group. The restriction enzyme map of each group is shown in Figure 2. Next, a group of plasmids containing pSGH1, which showed the strongest association with the synthetic DNA probe conducted in Example 3 and is thought to contain almost full-length cDNA,
In particular, for pSGH1, the entire nucleotide sequence of its translated region was determined using the Sanger method using M13 phage [Sanger et al., Proc. Natl. Acad. Sci, USA, 74 ,
5463 (1977): Amersham M13 cloning and
sequencing handbook]. The sequences are shown in Table 1. In Table 1, base numbers 1-66 code for the signal peptide, and base numbers 67-630 code for the mature peptide of chum salmon growth hormone. The amino acid sequence predicted from the cDNA sequence contained in pSGH1 completely matches the amino acid sequence near the N-terminus and near the C-terminus determined from chum salmon growth hormone peptide, and this cDNA encodes chum salmon growth hormone. This was confirmed.
Escherichia coli containing pSGH1, pSGH3, and pSGH8 (ESGH1, ESGH3, and ESGH8, respectively) were introduced in June 1981.
It has been deposited as FERM BP-551, 552 and 553 with the Institute of Microbial Technology, Agency of Industrial Science and Technology on the 23rd. [Table] Effects of the Invention According to the present invention, a recombinant plant incorporating DNA encoding a fish growth hormone polypeptide is produced.
DNA, a microorganism containing the recombinant DNA is obtained,
These can be used for mass production of fish growth hormone polypeptides.

[Brief explanation of the drawing]

FIG. 1 schematically shows cDNA synthesis by the Okayama-Berg method and the construction process of a recombinant plasmid containing the DNA. Figure 2 shows pSGH1, pSGH3,
A restriction enzyme map of cDNA contained in pSGH8 is shown.

Claims (1)

[Scope of Claims] 1. A DNA encoding a fish growth hormone polypeptide comprising the following peptide sequence. [Table] [Table] 2 Recombinant DNA incorporating DNA encoding a fish growth hormone polypeptide containing the following peptide sequence. [Table] [Table] 3. Recombinant DNA of Claim 2 named plasmid pSGH1, pSGH3 or pSGH8. 4. A bacterium containing recombinant DNA incorporating DNA encoding a fish growth hormone polypeptide containing the following peptide sequence. [Table] 5. The bacterium of claim 4, wherein the bacterium belongs to E. coli.