JPS61257999A - Prolactin polypeptide of fish - Google Patents

Abstract

NEW MATERIAL:A polypeptide having polypeptide sequence shown by the formula. USE:A bait for fish farming having growth promoting acting on fish and controlling action on fresh water adaptation ability. PREPARATION:Poly(A)RNA is separated from pituitary of Oncorhynchus keta by guanidium isothiocyanate cesium chloride method, it is blended with a vector primer and a reverse transcriptase, incubated, a complementary cDNA is synthesized and a cDNA-vector primer DNA is obtained. Then, it is incorporated with DNA ligase, DNA-polymerase and ribonuclease and a cDNA-containing recombinant DNA is subjected to ring formation to give a plasmid. Further, transformation of Escherichia coli is carried out by the use of it, Escherichia coli is cultivated in a nutritive medium, prolactin polypeptide of fish is accumulated in the culture mixture and separated.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a DNA encoding a prolactin polypeptide of fish, such as chum salmon, a recombinant DNA incorporating the DNA, a microorganism containing the recombinant DNA, and a microorganism containing the recombinant DNA. This invention relates to a method for producing fish prolactin polypeptide. Fish prolactin has a growth promoting effect in fish.

Furthermore, when adult chum salmon are transferred to fresh water, the plasma osmolarity decreases slightly after 24 hours, but remains constant after that, so adult chum salmon are known to have the ability to adapt to fresh water. In response to this decrease in osmotic pressure, blood prolactin concentration also decreases.
Since prolactin significantly increases after 24 hours, it is thought that prolactin plays an important role in the freshwater adaptation of chum salmon.

Therefore, the prolactin polypeptide provided by the method of the present invention is expected to have wide applications in the fish aquaculture industry.

BACKGROUND OF THE INVENTION Mammalian prolactin is produced in the pituitary gland, and its activity and structure are known.

For example, for human prolactin, J.B.

Endocrinology 71.209 by Jos imov ich et al.
(1962), Proceedings of the National Academy of Sciences (Proc. Natl.) by L. M. Sherwood et al.

Acad, Sci,) [ISA, 58.2307
(1967). There have been several reports on the isolation of prolactin from fish.

Isolation example from tilapia: S, +1. Farmer et al., General and Comparative Endocrinology (Gen, Camp, Bndocrin),
31.60-7H1977) Isolation example from salmon: D
, R. Idler et al., Gen. Camp.
Bndocrin, ), 35.40! J-418 (
1978) Isolation example from carp Go, B., Ng et al.
en.

Comp, 已ndocr+++) 42. 14
1-146 (1980) Isolation example from chum salmon: H9K
awauchi et al., General and Comparative Endocrinology ((ien, C0IIIGI,
Endocrin, ), 49.446-458 (1
983> On the other hand, regarding the mammalian prolactin gene, the raft prolactin gene (N, 8. Cooke
et al., Journal of Biological Chemistry (J.

Biol, Chem, ) 255. No, 13.
6502 (1980)) bovine prolactin gene (J
, HoNilson et al.: Nucleic Acids Res, 8.
Nα7゜1561 (1980)), human plactin gene (N, B, Cooke et al. Journal of Biological Chemistry (J, Biol)
, Chem, ) 256. Nc8.400? (19
81) ], but there are no reports yet on the prolactin gene in fish.

Problems to be Solved by the Invention Fish prolactin has the effect of promoting the growth of fish and regulating the ability to adapt to freshwater, so it is useful as a composition of fish feed. Quantity is limited. Therefore, it is desired to develop a method for supplying fish prolactin in large quantities at low cost.

Means for Solving the Problems The present inventors conducted research on a method for producing fish prolactin using recombinant DNA techniques. the result,
We succeeded in collecting NA complementary to fish prolactin polypeptide and producing recombinant DNA and microorganisms containing it, which can be used for fish prolactin production. That is, Metsenger R from the chum salmon pituitary gland.
Extract NA (mRNA) and extract complementary to it NA
(cDNA), then synthesize a DNA probe corresponding to the amino acid sequence near the N-terminus of chum salmon prolactin, and generate cDNA that hybridizes with this DNA.
By selecting A, the chum salmon prolactin gene was cloned, and the entire base sequence of its cDNA was determined. The present inventors conducted further research and found that by culturing microorganisms containing recombinant DNA that had incorporated DNA encoding chum salmon prolactin, a significant amount of chum salmon prolactin polybebutide accumulated in the culture. , we have completed the present invention.

The present invention will be explained in detail below.

The present invention provides fish prolactin polypeptides, particularly polypeptides having the peptide sequences shown in Table 1. The polypeptide can be produced using recombinant DNA techniques as described below.

That is, using fish prolactin mRNA as a template, DNA (cDNA) complementary to the mRNA is prepared, and a recombinant plasmid incorporating the cDNAΔ is prepared. Furthermore, the recombinant plasmid is inserted into a host microorganism. The DNA and recombinant plasmids can be used for the amplification of the prolactin gene, especially in bacteria such as Escherichia coli. A microorganism having the recombinant plasmid is useful for producing fish prolactin polypeptide in large quantities at low cost.

Therefore, the present invention provides a DNA encoding a fish prolactin polypeptide and a recombinant DNA incorporating the DNA.
A. A microorganism containing the recombinant DNA and a method for producing fish prolactin polypeptide using the microorganism are provided.

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) cell o-x (oligo(dT)cell
Polyadenylic acid [
RNA with poly(A)] Cpo'J (A) RN
A) Separate.

This boy! I (A) Using RNA as a template, double-stranded DNA is synthesized using reverse transcriptase. Recombinant is in vitro D
It is obtained by inserting the synthesized DNA into vector DNA such as E. coli plasmid DNA using NA recombination technology. DNA complementary to chum salmon brolactin mRNA
A recombinant plasmid containing A is selected based on the marker carried by the plasmid.

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. This frozen pituitary gland was treated with guanidium isothiocyanate (guanidium isothiocyanate).
iocyanate), crush, and solubilize. Then C5Cj! The solution layer is layered, and after ultracentrifugation, the total cytoplasmic RNA is obtained as a precipitate. Alternatively, only RNA can be precipitated and recovered by adding LiCβ to the guanidium isothiocyanate solubilized product.

The extracted RNA was dissolved in a high salt concentration (e.g. 0.5 M) solution of NaCl or KCl, and oligo(clT)
The mR containing poly(A) is passed through a cellulose column.
Adsorb NA onto the column. Water, 10mM Tris-HC
j! ! The mRNA with poly(A) is isolated by elution using a low salt solution such as buffer solution.

Below, Okayama-Berg (Okayama-Berg)
) method [Okayama Akira and Barb (Okayama
&Berg); Molecular & Cellular
Biology (Mol, Ce1l, Biol, )
2, 161 (1982)), cDNA
and its integration into a vector.

First, vector primers are synthesized. As a vector, for example, pCDVl can be dissolved in a suitable solution, such as Tris-H.
(l buffer (e.g. pf17, 5.10mM),
treated with Kpn I in a solution containing M g Cl1s (e.g. >6mM), NaCj! (e.g. 10mM),
Cut pCDVl at the Kpn 1 site.

This DNA was added to a Tris-HCl buffer (e.g. pH 6,
8,30mM), sodium cacodylate (e.g. 140mM), CoCC (e.g. 1mM), dithiothreitol (e.g. 0.1mM) and dTTP (e.g. 0.25mM) with terminal deoxynucleotidyl transferase at a constant temperature (e.g. 37°C). ) for a certain period of time (for example, 20 minutes) to add approximately 60 thymidyl residues to both 3' ends of the vector DNA. Furthermore, this DNA was added to Tris-H (
l buffer (e.g. pH 7, 5, 10mM), MgCl
t (e.g. 6mM>, NaCj! (e.g. 100
After cleavage with EC0RI in a solution containing nM), low melting point agarose gel electrophoresis was performed [Lar
s Wieslander ): 7f')
hemistry, ) house, 305 (1979)
, hereinafter referred to as LGT method], and a fragment of about 3.1 kilobases is recovered. The DNA is then dissolved in a high salt concentration (e.g. 0.5 M) solution of NaCl or KCl and passed through a PoIJ(dA) cellulose column to obtain poly(
Only vector primer molecules having T) are adsorbed onto the column. Elute with a low salt solution such as Lys-HC7 buffer (water, 10mM) to isolate only the vector brimer molecules attached with Bo'J (T).

Next, linker DNA is synthesized. For example pLIDNA
in a suitable solution, such as Tris-HCl buffer (e.g. pH 7, 5, 10mM), MgCl 1° (e.g. 6mM).
M), treated with PstI in a solution containing NaCl (eg 50mlJ) to cleave the PstI site of pLl.

This DNA was treated in the same manner as for vector primer synthesis except that dGTP was added instead of dTTP, and 1
Approximately 5 oligo (dG) chains are added. The DNA is dissolved in a suitable solution such as Tris-HCl buffer (e.g. pH
7, 5, 10mM), MgCj22 (e.g. 6mM).

NaCj! (e.g. 60mM) in a solution containing Hin
Cut at dI[[. DNA fragments of approximately 0.5 kilobases are fractionated by agarose gel electrophoresis and recovered using DEAE paper. In this way, linker DNA is obtained.

cDNA synthesis is performed using the po'J (A) RNA, vector primer, and linker DNA obtained as described above. Poly(A) RNA, vector primer DNA
A with Tris-H (l buffer (e.g. pH 8, 3, 50
mM), MgCRx (e.g. 3 mM).

KCj! (e.g. 30mM), dithiothreitol (e.g. 0.3mM), dATP, dTTP.

Reverse transcriptase is reacted in a solution containing dcTP and dGTP (for example, 2 mM each) at a constant temperature (for example, 37° C.) for a certain period of time (for example, 40 minutes).

At the 3' end of the RNA-DNA duplex thus obtained, dT
The vector primer except that TP is changed to dcTP (
Oligo(dC) was added under the same conditions as when adding dT) chains.
Add around 15 chains. This DNA is Tris-HCJ
2 buffer (e.g. pH 7, 5, 10mM), MgCβ
2 (for example 6n+M), NaCj! (For example, 6
cleaved with HindIII in a solution containing 0mM). The previously prepared linker DNA was mixed with this DNA, and a Tris-HCl buffer (e.g. pH 7, 5, 20mM) was added.
MgCL (e.g. >4mM, (NH4)2SO4
(eg 10mM).

K (1 (e.g. 0.1 M), β-nicotinamide adenine dinucleotide (β-NAD) (e.g. 0, 1
Incubate with E. coli ON^ligase in a solution containing m M ) for a certain period of time (for example, 16 hours) at a constant temperature (for example, 12° C.). In this way, cDNA and linker DNA are circularized. Add d to this reaction solution.
ATP, dTTP, dGTP.

Add dCTP to a final concentration of 40 μM, and add E. coli DNA! A recombinant plasmid containing a complete double-stranded cDNA is obtained by adding I gauze, E. coli DNA polymerase 11 and E. coli ribonuclease H to convert the RNA portion into DNA.

Using the thus obtained recombinant plasmid, Escherichia coli, for example, Escherichia coli C600SF3 strain, is grown using Scots.
tt) et al.'s method [Katsuya Shigesada: Cell Engineering 2.616 (1
983) ).

Since the ampicillin resistance gene is present on the recombinant plasmid obtained above, the transformed E. coli exhibits ampicillin resistance. The following procedure is commonly used to select from these ampicillin-resistant (Ap'') strains harboring novel recombinant plasmid DNA with a gene complementary to fish prolactin mRNA. The resulting transformed strain was immobilized on a nitrocellulose filter, and a synthetic N having a DNA sequence predicted from the known amino acid sequence of chum salmon brolactin was immobilized on a nitrocellulose filter.
associate with AAp-bu and select those that associate strongly [
Grunstein-H
Proceedings of the
National Academy of Sciences (Proc.
, Natl, Acad, Sci,), USAAl, n
.

3961 (1975)). Probe DNA was prepared using the usual triester method [Journal of American Chemical Society (J, Am, Chem, Sac, )]
, 97.

7327 (1975)). Synthetic NA
The selection by Ap-bu is Southern.
[Journal of Molecular Biology (J9Mol, Biol, ), 98.503]
(1975)], and by this method, recombinant plasmid DNA having a gene complementary to chum salmon prolactin mRNA can be identified.

An example of a recombinant plasmid obtained in this way is p
It is SPRL1. This plasmid can be used as a source of DNA encoding chum salmon prolactin.

The DNA is excised from a plasmid containing DNA encoding chum salmon brolactin, inserted into vector DNA, the resulting recombinant DNA is introduced into a microorganism, and the resulting transformant is cultured to produce chum salmon prolactin. Butide is produced and accumulated in the culture,
By collecting this, chum salmon prolactin polypeptide can be produced.

A suitable example of a plasmid containing DNA encoding chum salmon prolactin is the above psPRL1.

Any vector DNA can be used as long as the inserted DNA can be expressed in microorganisms. Preferably, a suitable promoter,
For example, tryptophan (trp), lactose (l
ac) system, PL system, etc., DNA can be inserted downstream, and the internal Shine-Dalgarno sequence (hereinafter abbreviated as SD sequence) and translation initiation codon (
ATG) is used in which the vector DNA is adjusted to an appropriate distance, for example, from 6 to 18 base pairs. A specifically suitable vector DNA is plasmid pG[! I can give Ll. pGELl is the plasmid shown in Figure 3, and the E. coli containing it was deposited as Bscherichia rGELl (FERM BP-629) at the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM BP-629) on October 6, 1981. ing. Recombination between the polypeptide-encoding DNA and the vector DNA is achieved by digesting both DNAs using restriction enzymes, and then recombining the T 4 DNA! This can be done using common recombinant DNA techniques using J gauze.

In the case of pSPRL1 and pGBL1 shown as specific examples, two stages of construction are performed as shown in FIG. That is, chum salmon prolactin mature peptide N from psPRll.
The 1aefff-Hlnd m fragment encoding the near terminus and the Hind m -Ban m fragment containing the remainder of the cDNA and a portion of the vector are obtained separately. on the other hand,
A synthetic NA linker as shown below is prepared.

The above DNA fragment and the synthesized NAU anchor are combined into T4DNA.
After ligation with ligase, the recombinant plasmid pSPRL^6 shown in FIG. 3 is obtained.

Next, chum salmon prolactin matures from pSPRL^6.
A Ban m-BamHI digested fragment encoding peptide is obtained, and a Ban 1-Bam11 I digested fragment containing a tryptophan promoter is obtained from pGBL1. The above two DNA fragments were joined with T4DNAU gauze to create the recombinant plasmid psPRLB shown in Figure 3.
Get 1. This plasmid has a region encoding mature chum salmon prolactin linked downstream of the tryptophan promoter.

The reaction conditions in the above recombinant technique are generally as follows.

The digestion reaction of DNA with restriction enzymes is usually 0.1 to 20
μg of DNA to 2-200mM (preferably 10-4
0mM) of trix-MCI (pH 6,0-9.5 preferably pH 7,0-8.0>, 0-200mM Na
CC2-30mM (preferably 5-10mM) Mg
In a reaction solution containing C1x, using 0.1 to 100 units of restriction enzyme (preferably 1 to 3 units per 1 μg of DNA) at 20 to 70°C (the optimal temperature varies depending on the restriction enzyme used), Do this for 15 minutes to 24 hours. The reaction is usually stopped by heating at 55 to 75° C. for 5 to 30 minutes, but a method of inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate can also be used.

Purification of DNA fragments generated by restriction enzyme digestion was performed using LG
This is performed using the T method, polyacrylamide gel electrophoresis, or the like.

The DNA fragment binding reaction is carried out using 2 to 200 mM (preferably 10 to 40 mM) Tris-HCI (pH 6, 1 to 9).
65, preferably pH 7.0-8.0), 2-20mM
(preferably 5-10mM) of MgCj! z, 0.1
~10mM (preferably 0.5-2.0mM)
of ATP, 1 to 50 mM (preferably 5 to lomM) of dithiothreitol, using 0.3 to 10 units of T4 DNA ligase at 1 to 37°C (preferably 3 to 20°C) for 15 minutes. ~72 hours (preferably 2-2
0 hours).

The recombinant plasmid DNA produced by the ligation reaction is
If necessary, the transformation method of Cohen et al.
Cohen, S.N. et al.: Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.
).

USA, 69.2110 (1972)) i: Yotte, introduced into E. coli.

Isolation of recombinant plasmid DNA from Escherichia coli containing recombinant plasmid DNA can be carried out by the method shown in Example 1 described below or by the method of Birnboim et al.
et al.: Nucleic Acid Research (Nucleic
Acids Res, ) Yu, 1513 (1979)
) etc.

After digesting plasmid DNA with 1 to 10 types of restriction enzymes, the cleavage site is examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis.

Furthermore, when it is necessary to determine the base sequence of DNA, the Maxam-Gilbert method [Proceedings of the National Academy of Sciences (Proc.
, Natl, Acad, Sci, ), 74
．． 560 (1977)) or the Sanger method using M13 phage [Sanga-(Sanga-(1977))]
ger) et al.: Proceedings of the National Academy of Sciences (Proc. Nat.
l, ^cad, Sci, ) USA.

74, 5463 (1977); Amar-Jam (A+n
M13 Cloning and Sea Fencing Node Book (cloning an
d sequencing handbook)).

The fish prolactin peptide of the present invention can be produced as follows.

That is, E. coli is transformed with -128BIOI using a plasmid (for example, psPRLB1), and E. coli having psPRLB1 is selected from ampicillin-resistant colonies. By culturing Escherichia coli containing psPRLB1 in a medium, fish prolactin polypeptide can be produced in the culture.

As the medium used here, either a synthetic medium or a natural medium can be used as long as it is suitable for the growth of E. coli and the production of fish prolactin polypeptide.

Carbon sources include glucose and fructose.

Lactose, glycerol, mannitol, sorbitol, etc. are nitrogen sources such as NH, (1°(NH4hS)
Other nutritional sources include K2HPO4, KH2
PO4, NaCj! .

Mg5O*, vitamin B+, MgCfa, etc. can be used.

Culture at pH 5.5-8.5. Culture is carried out at a temperature of 18 to 40°C by aeration and stirring.

Since chum salmon prolactin polypeptide accumulates in the cultured bacterial cells after 5 to 90 hours of culture, the bacterial cells are collected from the culture, treated with lysozyme, and then repeatedly frozen and thawed to crush the bacterial cells. A polypeptide is collected from the supernatant obtained by centrifugation according to a conventional polypeptide extraction method.

In addition, the detection of the polypeptide can be done by directly using Laemmli (L) cultured bacterial cells.
aemmli) sample buffer [Laemmli
mml i), Nature, 2
27, 680 (1970)), 5D
S-polyacrylamide gel [Laemm']
Method of li): carried out by Coomassie brilliant blue staining.

Examples of the present invention are shown below.

Example 1. Poly(Δ)RNA from chum salmon pituitary gland
Preparation: From the pituitary gland of chum salmon, the guanidium thiocyanate-cesium chloride method [Maniatis
) et al., eds., Molecular Cloning (Molecular Cloning)
lar Cloning), +1196. :l-
Co1d Spring + Harbor
Harbor); Katsuya Shigesada, Cell Engineering, 2,
616 (1983)) with poly(A)
A was prepared as follows.

2 g of frozen chum salmon pituitary gland (approximately 30 individuals) was mixed with 4M guanidinium thiocyanate), (1.5%-+f lucosine, 5mM sodium citrate (pH 7) and 0.IM
The mixture was crushed and solubilized in 10 IIll of a solution consisting of β-mercaptoethanol using a Teflon homogenizer (5 rpm). This homogenate was passed through an 18G syringe needle several times to disrupt the DNA. 5.7MCsC1,0,1M E
1.2 ml of each DTA (pH 8) solution was dispensed into ultracentrifuge tubes, and the homogenate was layered thereon. old tac
35.00Orpm with hi RPS 40 rotor
After centrifugation for 15 hours, RNA was collected as a precipitate. R
Precipitate the NA with Tris-HCj containing 1mM EDTA.
! (pH 8,0) solution in 10 ml of phenol-
After extraction with chloroform, it was recovered by ethanol precipitation. Approximately 1 mg of the obtained RNA was mixed with 10 mM) Lis-HCj!
(pH 8.0) and 1 ml of a solution consisting of 1 mM EDTA.

Incubate at 65°C for 5 minutes, Q, 1 ml of 5MN
a(l) was added. The mixture was diluted with oligo(dT) cellulose.
Column (manufactured by P-L Biochemicals)
The mixture was subjected to 07 toography (column volume 0.5 ml). mRNA with adsorbed poly(A) (10mM) Lis-HCj! (pH 7,5) and 1mM EDT
It was eluted with a solution consisting of A and Q, and fractionated into 2 ml portions. 3～
Collect both parts of the 5th and mRN with Po'J (A)
About 10 μg of A was obtained.

Example 2. CDNA synthesis and insertion of the DNA into a vector: Okayama-Berg's method [Molecular and Cellular Biology (Molecular and Cellular Biology)]
Mol, Ce11. Biol, ), 2, 16
cDNA was synthesized and a recombinant plasmid incorporating it was constructed according to H1982). An outline of the process is shown in FIG.

pcDVl (Okayama and Barb)
-ma & Berg): Molecular Pot and Cellular Biology (Mo1. Ce1l, Ria
l, ), 3.280 (1983) ) 400 p.
g to 10mM) Lis-HCj! (pH 7,5), 6 m
M M g CR2 and 10 mM NaC (l) plus 500 units of Kpn
I (manufactured by Takara Shuzo Co., Ltd.; unless otherwise specified, all restriction enzymes are manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was reacted at 37°C for 6 hours to cleave at the Kpn■ site in the plasmid. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation.

Approximately 200 μg of the Kpn I-cleaved DNA was mixed with 40 mM sodium cacodylate and 30 mM Tris-HCl (pH 6).
, 8), add dTTP to 0.25 mM to 200 μl of a buffer solution (hereinafter abbreviated as T d Tit!i solution) consisting of 1 mM CaCl2 and 0.1 mM dithiothreitol (hereinafter abbreviated as DTT). In addition,
In addition, 81 units of terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT) (P
-L Biochemicals), 37
℃ for 11 minutes. Here, Kpn of pCDVI
Approximately 67 poly(dT) chains were added to the 3' end of the I cleavage site. Phenol-chloroform extraction from the solution,
pC with poly(dT) chains added by ethanol precipitation
Approximately 100 μg of DVI DNA was recovered. 10 of the DNA
mM) Lis-HCf (pH 7,5), 6mM Mg
CL, 100mM NaCJ! A buffer solution 150 consisting of
In addition to μβ, 360 units of EcoRI were added and 3
The reaction was carried out at 7°C for 2 hours. After treating the reactant with the LGT method,
A DNA fragment of about 3°IKb was recovered, and about 60 μg of poly(dT)-added pCDV1 was obtained. The DNA was 1QmM
) Squirrel-HCj! (pH 8,0) and 1mM ED
Solution consisting of TA 500J11! After incubating at 65°C for 5 minutes, cool on ice and add 5M N of 50III.
aCJ was added. The mixture was subjected to oligo(dA) cellulose column chromatography (manufactured by Collaborative Research). Poly(dT) with sufficient chain length is adsorbed onto the column, and this is converted into lQmM)lith-HCj2 (p)I8
．． Elute with a solution consisting of 0) and 1mM EDTA. 27 μg of pcDVl (hereinafter abbreviated as vector primer) to which J (dT) chain was added was obtained.

Next, linker DNA was prepared.

pLl (Okayama and Barb)
& Berg): Molecular and Cellular Biology (Mo1.Ce11.Bi>, a
, 280 (1983)) approximately 14 μg in 10 mM) Sux-HCl (pH 7,5).

6mM MgCj! 2 and 50mM NaCj!
In addition to 200 μl of buffer consisting of
stI was added and reacted at 37°C for 4 hours to cleave pLIDNA at the pstr site. The reactant is converted into phenol-
After chloroform extraction, ethanol precipitation was performed, and PstI
Approximately 13 pg of pLIDNA was recovered. The DN
Approximately 13 μg of A was added to a TdT slow solution at a final concentration of 0.25 mM (7
) In addition to 50 μβ of a solution containing dGTP, T d
54 units of T (manufactured by P-L Biochemicals) were added and incubated at 37°C for 13 minutes to add approximately 14 (dG)611 units to the 3' end of the pstt cleavage site of pLl. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation.

100pl of the DNA! 10mM) of Lis-HC1 (p
H7,5), 6mM MgCj! 2 and 60mN
acj! A buffer solution consisting of 1001! In addition to 80
One unit of HindllI was added, incubated at 37°C for 3 hours, and pLIDNA was cleaved at the Hindlll site. The reaction product was fractionated by agarose gel electrophoresis, and approximately 0
．． A 5 Kb DNA fragment was extracted using the DEAE paper method (Dretze et al., Analytica).
.

112, 295 (1981)) and oligo (
dG) Chained linker-DNA (hereinafter simply referred to as linker-D)
(abbreviated as NA) was obtained.

Approximately 2 μg of poly(A) RNA prepared above and approximately 1.4 μg of vector primer were added to 50 mM) Lis-HCj! (
pH 8,3), 8mM MgCL, 30mM
KCj! , 0.3mM DTT, 2mM dNTP (
dATP, dTTP, dGTP and dCTP) and 10 units of glue bonuclease inhibitor (P-L
Solution 22.
Dissolved in a 3μ solution, added 10 units of reverse transcriptase (manufactured by Seikagaku Corporation), and incubated at 37°C for 40 minutes.
NA complementary to Δ was synthesized. The reaction product was extracted with phenol-chloroform and precipitated with ethanol.
- Vector brimer DNA with added DNA double strands was recovered. The DNA was treated with 66pM dCTP and 0.2μ
gpo! J (A) was dissolved in 20 μl of TdT buffer containing 14 units of T d T (P-L Bioche
(manufactured by Micals) and incubation was performed 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 the (dC)I-added cDNA-vector primer DNA was recovered by ethanol precipitation. MDNA (10mM) x-HCj! (pH 7.5>, 6mM MgC1, and 60mM NaCj!), 20 units of HindIII was added, incubated at 37°C for 2 hours, and cleaved at the HindI11 site. The reaction product was extracted with phenol-chloroform, and precipitated with ethanol. Q, 5 pmole of (dC) conjugated cDNA-
Vector primer DNA was obtained. The DNA0.081
)+l1ole and the linker-DNA0.16 above.
pmole lQmM) Lis-HC1 (pH 7,5),
0. Dissolved in 40 μl of a solution consisting of IM NaCβ and 1 mM EDTA at 65°C.

The cells were incubated at 42°C and 0°C for 10 minutes, 25 minutes, and 30 minutes, respectively. 20mM) Lis-H (1 (pH 7,5
), 4mM MgCL, 10mM (NH4)2
SO4,0,IM KCl and 0.1 m Mg-
A reaction solution was prepared with a total volume of 400 μl based on the composition of NAD. 10 units of Escherichia coli DNA U gauze (New [manufactured by England Biolabs) was added to the reaction solution, and the mixture was incubated at 11° C. overnight. Components were added to the reaction solution to give 40 μM each of dNTP and 0.15 mM β-NAD, and 5 units of E. coli DNA'l gauze and 7 units of E. coli DNA polymerase I (P-L
Bio-chemicals) and 2 units of E. coli ribonuclease H (P-L Biochemicals)
ls) and incubated at 12°C and 25°C for 1 hour each.

In the above reaction, circularization of recombinant DNA including cDNA,
The RNA portion of the RNA-DNA duplex was replaced with DNA, producing a complete double-stranded DNA recombinant plasmid.

Example 3. Selection of recombinant DNA containing chum salmon brolactin cDNA: Using the recombinant plasmid obtained in Example 2, E. coli C60
0SF8 stocks [turtle C1:/ ((:ameron):
Proceeding of the National Academy of Sciences (Proc. Natl.
Acad, Sci, ) USA.

72.3416 (1975)) by the method of 5Cott et al. [Katsuya Shigesada: Cell Engineering, 2.616 (1983)
). Approximately 1,700 colonies obtained were immobilized on nitrocellulose. Synthetic NA corresponding to the 122nd to 127th amino acid sequence from the N-terminus of chum salmon brolactin, i.e. (L) (The 6th base is A, T, G, or C, and the 7th base is A or G.

The 9th base is T or C1, and the 12th base is A or G, which together form a mixture of 32 synthetic NAs. ) strongly associated with the 32p-labeled probe at 42°C.
The selected strain was Grunstein Hognes (Gruns).
tein-Hogness), Proceedings of the National Academy of Sciences (Proc, Natl, ^cad, Sci,) U
SA, 72.3961 (1975)). obtained 7
Regarding bacterial strains, Southern method [Journal of Molecular Biology (J8M)
ol, Biol, ), 98, 503 (1975)
), the association with the above probe was confirmed. These plasmids are pSPRL1, 2.4.7°8, 1
0. 11, and since both had DNA sequences predicted from the amino acid sequence of chum salmon brolactin, they were thought to contain prolactin cDNA.

Example 4. Base sequence of plasmid psPRL1: The seven plasmids obtained above were digested with various restriction enzymes, and the cleavage map of the cDNA portion was determined. plasmid p
A restriction enzyme map of the cDNA in SPRL1 (hereinafter referred to as 5PRLI) is shown in FIG.

Next, regarding pSPRL1, which showed the strongest association with the synthetic NA probe conducted in Example 3 and is thought to contain almost full-length cDNA, the entire nucleotide sequence of its translated region was analyzed using Sanger (Sange) using M13 phage.
r) Law (Sanger et al.: Proceedings of the National Academy of Sciences (Pro
c, Natl, Acad, Sci,), t
lsA.

74, 5463 (1977): Amersham M13 cloning and sequencing
Determined according to handbook). The nucleotide sequences are shown in Table 1. In Table 1, base numbers 1-69 code for the signal peptide, and base numbers 70-630 code for the mature peptide of chum salmon prolactin.

The amino acid sequence predicted from the cDNA sequence contained in pSPRL1 completely matches a part of the amino acid sequence determined from natural chum salmon prolactin.
It was confirmed that A encodes chum salmon prolactin.

Escherichia coli containing pSPRL1 is ε5cherichia
coli8SPRL1 (FεRM BP-776)
As of April 26, 1985, it was deposited with the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology.

(Left below) Table Example 5. Construction of a recombinant plasmid encoding chum salmon brolactin: (1) Construction of a recombinant plasmid ρ5PRLBI encoding mature chum salmon brolactin from pSPRL1. HCII
(pH 7.5), 7mM MgClw, and 100mM NaCl (hereinafter abbreviated as Y-100 buffer).
30 units of I (manufactured by Toyobo Co., Ltd.) were added, and a digestion reaction was carried out at 37°C for 2 hours. Using the LGT method, pSPR was obtained from the reaction solution.
Approximately 0.7 Kb DNA containing the N-terminal side of prolactin encoded by L1, the right 5' untranslated region, and a portion of the vector
About 1 rope of fragments was obtained. This Q, 7Kb DNA is Y-10
0 buffer, 8 units of restriction enzyme HaeI was added, and a digestion reaction was carried out at 37°C for 2 hours. After this reaction solution was subjected to polyacrylamide gel electrophoresis, a 43 bp DNA fragment corresponding to the vicinity of the N-terminus was obtained by glass filter filtration.
I got 1■.

Next, add 211 g of pSPRL1 to 30 g of Y-100 buffer.
A Banma unit and 116 units of HindII were added, and a digestion reaction was performed at 37°C for 2 hours.

About 1.0 x g of a DNA fragment of about 3.7 Kb containing the C-terminal side of chum salmon brolactin encoded by pSPRL1, the 3'-untranslated region, and a portion of the vector was obtained from the reaction solution by the LOT method.

On the other hand, the following D
A N A IJ linker was synthesized.

First, 1g+++er and 15mar of wood-like DNA were prepared using the usual triester method [Crea, R. et al.: Proceedings of the National Academy of Sciences (Proc., Natl., Aca.
d.

Sci, >, tlsA, 25.5765 (1978)
:] was synthesized.

13mer and 16mer Ichiki DNA 4Qp each
mole 50mM) Lis-HC1(+)H7,5)1
0mM MgC12, 10mM DTT and 1mM
Dissolve in a solution containing ATP for 20 minutes, add 6 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), and incubate at 37°C for 30 minutes.
The phosphorylation reaction was performed for 0 minutes.

Haem-Hind■ fragment derived from psPRL1 obtained above (43 bp) o, o 8xmole, BanI
II-HlndI[[fragment (approximately 3.7 Kb) 0
．． 02 pmole 50mM') Lis-HCj2
(pH 7,5>, 10mM MgC12, 10mM D
Dissolved in solution 30 relative containing TT and Id ATP,
To this was added 5 g of the above synthetic NAIJ oxidation reaction solution. Six units of T4DNΔ ligase (manufactured by Takara Shuzo Co., Ltd.) were added to this mixture, and a binding reaction was performed at 4° C. for 18 hours. E. coli strain 294 was transformed using the reaction mixture to obtain Apl colonies, and plasmid DNA was recovered from these colonies to obtain psPRL86 shown in FIG. psPRL
The structure of A6 is PstI, BamHI.

It was cut with coRI, Banm, BglII, and old ndIn, and confirmed by agarose gel electrophoresis.

(2) Integration of the region encoding mature chum salmon prolactin of psPRLA6 into the expression vector pGEL1:
psPRLA6 2x 3011t! Y-100 buffer, 8 units each of BamH1 and Ban I were added, and a digestion reaction was performed at 37°C for 3 hours. LG from the reaction solution
By the T method, about 0.1 g of a DNA fragment of about 1.2 Kb encoding the entire mature chum salmon brolactin was obtained.

Separately, 2 tails of pGBLl were dissolved in 40JJII Y-100 buffer, and 9xmHI and Ran m were added at 10% each.
Units were added and a digestion reaction was carried out at 37°C for 3 hours. From this reaction solution, about 0.2 g of a DNA fragment of about 2.7 Kb containing a tryptophan promoter was obtained by the LGT method.

About 1.2 Kb 0.01 g of the Ran m -Ram old fragment of pSPRLA6 obtained above and 13a of pGEl, l
n gl-BamHI fragment (approximately 2.7 Kb) 0.0
15xg in 50mM Tris-HCl <98'? , 5
), 10mM MgCl2, dissolved in 20 scraps of a solution containing 10dDTT and ldATP, T4DNl
Six units of gauze were added and the binding reaction was carried out at 4°C for 18 hours.

Using the reaction solution, Escherichia coli strain 294 was transformed and A p
1 colony was obtained, and this colony's plasmid DNA
A was collected to obtain psPRLB1 shown in FIG. p
The structure of sPRLBl is BcoRI.

tlindI[[, 13an[, pst l, Bam
It was cut with HI and confirmed by agarose gel electrophoresis.

Example 6. Production of chum salmon prolactin peptide by Escherichia coli containing psPRLBl: Using the recombinant plasmid psPRLBl obtained in Example 5, E. coli W 3110strA strain (FBRM
BP-732) was transformed. The obtained Apl colony was transferred to 3 ml of MCC medium [0.6% Na2HPO4,
0.3%KH2PO, 0.5%NaCj2, 0.1%
NH<ci, o, 5% glucose, 0.5% casamino acids, 1+++lJ MgSO4°4g/ml vitamin 7B+, 11)17.2) and cultured at 30°C for 18 hours. The obtained culture solution was g, oo.

The cells were collected by centrifugation at rpm for 10 minutes. After suspending this bacterial cell in Laemmli sample buffer, 5D
S-polyacrylamide gel electrophoresis was performed and stained with Coomassie brilliant blue, and the molecular weight was approximately 27.00.
A polypeptide band was detected at the 0 site. This band was not present when E. coli not containing the plasmid was used. As a result, it was found that Escherichia coli harboring psPRLB1 produced a large amount of chum salmon prolactin polypeptide.

Escherichia coli containing psPRLBl is Bscherichi
a coli Snake SPRLBI (FERM BP-777
) and was deposited with the Institute of Fine Technology on April 26, 1985.

Effects of the Invention According to the present invention, there is provided a recombinant DNA incorporating DNA encoding a fish prolactin peptide.

Microorganisms containing the recombinant DNA are obtained, and these can be used for mass production of fish prolactin polypeptides.

[Brief explanation of the drawing]

Figures 1 (1) and (2) outline the process of cDNA synthesis by the Okayamberg method and the construction of a recombinant plasmid containing the DNA. FIG. 2 shows a restriction enzyme map of the cDNA encoding Shirozagabrolactin contained in ρ5PRLI. Figure 3 shows recombinant plasmid psPRLA6. psP
The construction process of Rl, 81 is shown. Figure 1 Bekkufu Rymer DNA rm mRNA AAaA -----+++++++-++ Procedural correction spontaneously) February 7, 1985

Claims (9)

[Claims] (1) Fish prolactin polypeptide having the peptide sequence shown in Table 1 (2) Fish prolactin is found in herring (Clupeif).
2. The polypeptide according to claim 1, which is a hormone having a growth-promoting effect on the growth of C. ormes. (3) D encoding fish prolactin polypeptide
N.A. (4) D according to claim 3, in which the fish prolactin polypeptide has the peptide sequence shown in Table 1.
N.A. (5) A recombinant DNA incorporating DNA encoding a fish prolactin polypeptide having the peptide sequence shown in Table 1. (6) The recombinant DNA according to claim 5, named plasmid pSPRL1. (7) A microorganism containing a recombinant DNA incorporating DNA encoding a fish prolactin polypeptide having the peptide sequence shown in Table 1. (8) The microorganism according to claim 7, wherein the microorganism belongs to Escherichia coli. (9) D encoding fish prolactin polypeptide
Fish prolactin, characterized in that a microorganism containing a recombinant DNA incorporating NA is cultured in a nutrient medium, a fish prolactin polypeptide is accumulated in the culture, and the polypeptide is collected from the culture. Method for producing polypeptides.