JPS62234096A - Prolactin polypeptide derivative of fish or such - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

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

When adult chum salmon are transferred to freshwater, plasma osmolarity decreases slightly after 24 hours, but remains constant after that, and adult chum salmon are known to have the ability to adapt to freshwater. In response to this decrease in osmotic pressure, blood prolactin concentration also increases significantly after 24 hours, and therefore prolactin is thought to play an important role in the freshwater adaptation of chum salmon.

Therefore, the prolactin polypeptide derivative 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 (endocrinology) by Josimovich et al.
Endo-crinolog V) 71.209 (1
962), Proceedings of the National Academy of Sciences (Proc. Natl.

^cad, Sci,) USA, 58.2307 (
It was reported in 1967>. There have been several reports on the isolation of prolactin from fish.

Isolation example from tilapia: S, W, Farmer
General and Comparative Endocrinology (Gen, Comp)
n, ), 31. 60-7H1977) and J
, 5pecker et al., Proceedings of the National Academy of Sciences (P.
roc, Natl, Acad, Sci,) USA
, 82.7490-7494 (1985) Isolation example from salmon: D. R. Icller et al., General and Comparative Endocrinology (Gen, Comp, Endocrin.), 3
5.409-41g (1978) Isolation example from carp:
T, B, Ng et al. General and Comparative Endocrinology (Gen.

(:omp, ndocrin,)42. 141
~146 (1980) Isolation example from chum salmon: H, K
Awauchi et al., General and Comparative Endocrinology (Gen, Camp,
Endocrin, ), 49,446-458 (198
3) Regarding the fish prolactin gene, the present inventor isolated the chum salmon brolactin gene, determined the structure, and
Prolactin polypeptide was also produced using engineered DNA techniques (Japanese Patent Application No. 60-99094).

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. Quantities are limited, and production by the aforementioned recombinant DNA techniques also results in low yields. Therefore, it is desired to develop a method for supplying a substance having fish prolactin activity at low cost in large quantities.

Means for Solving the Problems The present inventors conducted research on a method for producing a substance having fish prolactin activity using recombinant DNA techniques. As a result, we succeeded in producing a recombinant DNA and a microorganism containing NA complementary to a fish prolactin polypeptide derivative, which can be used to produce a fish prolactin derivative. The present inventors discovered that a significant amount of chum salmon prolactin polypeptide derivatives accumulated in this product, leading to the completion of the present invention.

The present invention will be explained in detail below.

The present invention provides a recombinant DNA incorporating DNA encoding a fish prolactin polypeptide derivative, a microorganism containing the recombinant DNA, and a method for producing a fish prolactin polypeptide derivative using the microorganism.

The recombinant plasmid of the present invention is prepared by the following general procedure.

DNA encoding a fish prolactin polypeptide is converted to DNA encoding a fish prolactin polypeptide derivative by various enzyme treatments, this is incorporated into vector DNA, and the resulting recombinant DNA is introduced into a microorganism. By culturing the transformant, a fish prolactin polypeptide derivative is produced and accumulated in the culture, and by collecting the derivative, a fish prolactin polypeptide transductor can be produced.

The origin of DNA encoding fish prolactin polypeptide is, for example, the DNA encoding chum salmon prolactin polypeptide cloned by Kuwana et al.
Plasmid psPRL1 containing A (patent application Sho 6O-990
94) and derivative plasmids thereof can be used.

E. coli containing plasmid psPRL1 is Escher
ichiacoliε5PRI, 1(FERM Of'
-776) on April 26, 1985, at the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology.

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 pTrs20 (patent application 1986).
-23319 and pKYP26. Escher ich containing pKYP26
iacoli I KYP 26 (FERλl
BP-863) and was deposited with the Institute of Fine Technology on August 8, 1985.

Specifically, DNA encoding a prolactin polypeptide derivative is obtained by the following method. That is, plasmid psPRL1 was cleaved by Kpn I treatment, Ba131
Digestion with Bam1 (I) and cleavage with Bam1 (I) were carried out for approximately 1.
2. DNA encoding the prolactin polypeptide of b.
Obtain a DNA fragment containing.

Separately plasmid l]KYP26[]amfl I and P
By cutting with stI, a DNA fragment of approximately 1.700 bp containing a portion of the lpp terminator is obtained. Furthermore, plasmid pTrs20 was cut with Sac I,
Blunt end formation by DNA polymerase I and Pst
A DNA fragment of about 1,000 bp containing a tryptophan promoter is obtained by cutting with I.

The resulting three DNAs were combined using T4 DNA IJ gauze to prepare a recombinant plasmid, which was used to transform Escherichia coli HBIOI to obtain ampicillin-resistant (Apl) colonies, from which plasmid DNA was recovered. The resulting plasmid pSPRL port 2
The restriction enzyme cleavage cuts for pSPRLQ52 and pSPRLQ52 are shown in FIG.

Plasmid psPRLl contains this [! 5cher
ich 1 acoli E S P RL 1 (
FIERM 0P-776) to Birnboim et al.'s method [Nucleic Acid Research (Nucleic
Ac1ds Res, ) 7, 1513 (1979)
"It can be collected by J.

Plasmid pKYP26 contains Escheri
chiacoli I K Y P 26 (FBR
MBP-863) by the method of Bernpoim et al.

pTrs20 can be produced by the method shown in Reference Example 1.

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
t, tg DNA at 2-200mM (preferably 10-200mM)
40mM) Tris-H (1 (pH 6.0-9.5 preferably pH 7.0-8°0), 0-200mM Na
Cj! , 2-30mM (preferably 5-10mM) MgCj! In a reaction solution containing 2, restriction enzyme 0.1
~100 units (preferably 1 to 1 Pg of DNA)
3 units) at 20 to 70°C (the optimum temperature varies depending on the restriction enzyme used) 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 the DNA fragments generated by restriction enzyme digestion was carried out using low melting point agarose gel electrophoresis (LGT method) [L, Wieslander: 7 Analytical Biochemistry (L, Wieslander)].
ioche+n1stry) 98, 305 (197
9)) or polyacrylamide gel electrophoresis.

The DNA fragment binding reaction is carried out using 2 to 200 mM (preferably 10 to 40 mM) Tri:X, -HCl (pH 6,1
~9.5, preferably pH-7.0~8.0), 2~
20mM (preferably 5-10mM) tVIgcL
, 0.1-10mM (preferably 0.5-2, Om
M) ATP, 1-50mM (preferably 5-1
T4 in a reaction solution containing 0mM) dithiothreitol.
Using 0.3 to 10 units of DNA ligase at 1 to 37°C (
Preferably at 3 to 20°C) for 15 minutes to 72 hours (preferably 2 to 20 hours).

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

[ISA, Ken, 2110 (1972)] into E. coli.

Isolation of recombinant plasmid DNA from Escherichia coli containing recombinant plasmid DNA is carried out by the method of Birnboim et al. [H.C.

Birnboim et al.: Nucleic Acids Res.
Yu, 1513 (1979)).

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 [Sanger
er) et al.: Proceedings of the National Academy of Sciences (Proc. Nat.
l, Acad, Sci,) USA.

74, 5463 (1977); Ame Jam (Ame
M13 Cloning and Sea Fencing Handbook (cloning and sea fencing)
sequencing handbook).

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

That is, using a plasmid (e.g. pSPRL028 or pSPRLQ52) to inject -128BIO into E. coli.
E. coli having psPRl, 02B or pSPRLQ52 is selected from ampicillin-resistant colonies. pSPRL028 or pSP
By culturing E. coli having RLQ52 in a medium, a fish prolactin polypeptide derivative 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 production of Escherichia coli raw material and fish prolactin polypeptide derivatives.

Carbon sources include glucose and fructose.

Lactose, glycerol, mannitol, sorbitol, etc. are nitrogen sources, but NH, Cj! .

Other nutritional sources include (NH,)2SO, casamino acids, yeast extract, polypeptone, meat extract, batatotributone, and corn steep liquor.
PO4, KH2PO4, NaCj! .

MgSO4, vitamin B,, MgC! x 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.

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

In addition, the polypeptide can be detected directly by using REM'J in cultured bacterial cells.
(Laemmli) sample buffer [Laemmli (L
ae+nml i), naichi+ -(Nature),
227, 680 (1970)) and then heated and dissolved in 5DS-polyacrylamide gel [Rem! j (La
Coomassie Brilliant Blue staining.

Examples of the present invention are shown below.

Example 1 Construction of chum salmon prolactin voripeptide derivative expression plasmid: DN encoding chum salmon prolactin polypeptide
61℃g of plasmid psPRL1 containing A was mixed with 50 pieces of 10mM) Sox-HCl2 (pH 7,5), 7mM
g Dissolve in a solution containing C12 and 6mM mercaptoethanol (hereinafter abbreviated as Y-0 buffer) and add 15 units of restriction enzyme Kpnl (manufactured by Takara Shuzo Co., Ltd.; unless otherwise specified, restriction enzymes manufactured by Takara Shuzo Co., Ltd. were used). ,37℃,
Digestion reaction was carried out for 3 hours. 10mM) Lis-HCj2
(pH 7.5>, add an equal volume of phenol saturated with 1mM EDTA, stir vigorously, and then perform low-speed centrifugation (3.
The aqueous layer was collected at 30 rpm for 10 minutes under the same conditions (hereinafter this process will be abbreviated as phenol extraction).
. Next, an equal amount of chloroform was added, and after stirring vigorously, an aqueous layer was collected by low-speed centrifugation (hereinafter, this treatment will be abbreviated as chloroform extraction). Add 1/10 volume of 3M sodium acetate, add 2.5 volumes of ethanol, and -20
After standing for 2 hours at ℃, refrigerated centrifugation method (4℃, 11,
000rpltl for 10 minutes) to collect the precipitate (hereinafter, this treatment will be abbreviated as the ethanol precipitation method).

This precipitate was mixed with 20mM) 111:1 pH 8,0
), 12mM CaClx, L 2mM MgCL
, dissolved in 50 jdl of a solution consisting of 200 mM NaCl and 1 mM EDTA, and incubated at 20°C for 5 minutes. BAL31 nuclease [Bethesda Research Laboratory-x (Bethesda Re5earchL)
aboratories)] was added, and 20
The decomposition reaction was carried out at ℃ for 20 minutes. After the reaction, phenol extraction and chloroform extraction were performed, and the DNA was extracted by ethanol precipitation.
was recovered. This DNA was mixed with 10mM Tris-MCI (
pH 7,5), 7mM MgCl2.100mM
A solution consisting of 6 mM mercaptoethanol (hereinafter abbreviated as Y-100 buffer) was dissolved in 4 (btl!), added 10 unit of restriction enzyme BamHI, and incubated at 37°C for 2 hours.
A time digestion reaction was performed.

From this reaction solution, about 0.5 μ of a DNA fragment of about 1.2 kilobase pairs (Kb) containing DNA encoding chum salmon prolactin polypeptide was obtained by the LGT method.

Separately, 3 g of pKYP26 was added to 404 Y-10
8 units of restriction enzyme BamHI dissolved in 0 buffer, Pst
10 units of I was added and the digestion reaction was carried out at 37°C for 2 hours. From this reaction solution, 0.311 g of a DNA piece of about 1.700 base pairs (bp) containing a part of the terminator of the /I)l) system was obtained by the LGT method.

Apart from these, 3■ pTrs20 and 40ρ Y-0
Dissolve in buffer solution, add 10 units of restriction enzyme Sac I,
Digestion reaction was carried out at 37°C for 2 hours.

After phenol extraction and chloroform extraction, DNA was recovered from this reaction solution by ethanol precipitation.

This DNA was added to 5QmM) Lis-HC1 (pH 7.5).
, 5mM MgCl2.10mM mercaptoethanol and 0.4mM dATP, dcTP.

30tIf of a solution consisting of dcTP and dTTP! Dissolve in the solution, add 4 units of DNA polymerase (manufactured by Takara Shuzo Co., Ltd.),
The reaction was carried out at 20°C for 1 hour. After phenol extraction and chloroform extraction, DNA was recovered from this reaction solution by ethanol precipitation. Dissolve this DNA in 304 Y-100 buffer, add 6 units of restriction enzyme PstI, and incubate at 37°C.
Digestion reaction was carried out for 2 hours. From this reaction solution, about 1000b containing the tryptophan promoter was extracted using the LOT method.
A 0.1 m DNA fragment of p was obtained.

The psPRL1-derived DNA fragment obtained above (approximately 1,20
0bp) approximately 0.2■, pKYP26-derived DNA fragment (
Approximately 0.05J1g of pTrs20-derived DNA fragment (approximately 1.0OObp) was
0mM Tris-HCl (pH 7,5), 10mM
MgCl2. 10mM dithiothreitol and 1mM
A solution containing M ATP (hereinafter referred to as T 4 D N A I
J gauze buffer) was dissolved in 30 ρ, 3 units of T4 DNA IJ gauze (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was incubated at 4°C for 1 hour.
The binding reaction was carried out for 6 hours.

E. coli HBIO1 [Polliver et al., Gene, 2.75 (1977)] was transformed using the reaction mixture to obtain colonies of Ap', and from these colonies, the method of Birnboim et al.
Nucleitsk and Research, 7.

1513 (1979)), and pSPRLQ28 and pSPR shown in Fig.
LQ52 was obtained. When the base sequences of these plasmid DNAs were determined, pSPRLQ28 was found to lack the DNA encoding the 4th amino acid from the N-terminus of chum salmon prolactin polypeptide, and the 5th amino acid ( It was revealed that the structure encodes a prolactin polypeptide derivative with pTrs20-derived methionine attached to the N-terminus of the part after (aspartic acid). Similarly, pS
PRLQ52 has a structure in which the DNA encoding the 9th amino acid from the N-terminus is deleted, and it encodes a prolactin polypeptide derivative with pTrs20-derived methionine attached to the N-terminus after the 10th amino acid (alanine). It became clear that.

Tables 1 and 2 show pSPRLQ28 and pSP
The base sequence of the chum salmon prolactin polypeptide derivative gene of RLQ52 and the amino acid sequence of the polypeptide derivative are shown.

Many 1 person-Genetic Code CUniuersal
Table 2 - Genetic Code CUniversal Example 2 ρ5PRLQ28 or ρ5PRLQ52
Production of chum salmon prolactin derivative polypeptide by Escherichia coli containing: Escherichia coli W31 by a conventional method using the recombinant plasmid pSPRLΩ28 or pSPRLQ52 obtained in Example 1.
10 plate strain (FERM 8P-732) Total transformation. ? + UfL Ap' colonies were treated with M containing 50ttg/ml ampicillin and 25g/ml tryptophan.
CG medium [0.6% Na 2 HP Os, 0.3%
K82P 04.0.5%NaCl1,0,1%NH
, C1, 0.5% glucose, 0.5% casamino acids, 1
mM MgSO4-44/m! Vitamin B+, pH
7,2) and cultured with shaking at 30°C. O.D. 0
becomes 1.0, add indole acrylic acid to 20
x/ml, and further cultured with shaking at 30°C for 3 hours. (The asked culture solution is 8.OOOrpm, 1
The cells were collected by centrifugation for 0 minutes. After suspending the cells in Laemmli's sample buffer, 5DS-polyacrylamide gel electrophoresis was performed and stained with Coomassie brilliant blue. A polypeptide band was detected at a site with a molecular weight of approximately 25,000.

This band was not present when E. coli not containing the plasmid was used. In addition, after 5DS-polyacrylamide gel electrophoresis, the method of Towbin et al.
, H,, et , S,, Proc, Natl
, Acad.

Sci, USA, 76, 4350-4354 (1
Western blotting was performed using 979), and polypeptides of about 26,000 and 25,000 of the above molecules reacted specifically with the anti-rhozoic brolactin antibody.

As a result, it was found that E. coli harboring pSPRL028 or pSPRLQ52 produced a large amount of chum salmon prolactin polypeptide derivatives.

The Escherichia coli strain containing ρ5PRLQ28 and pSPRLQ52 is Escherichia coli ESPRL
Q28 (FERM 0P-1006) and Esche
richia coli ESPRLΩ52 (FERM
BP-1007) and was deposited with the Institute of Fine Technology on March 28, 1986.

Example 3 After culturing chum salmon prolactin polypeptide derivative-producing bacteria according to Example 2, 8. OOOrpm, centrifuge for 10 minutes to collect bacteria, and add 30mM NaCj! and 3QmM
) It was washed with a buffer solution (pH 7.5) containing Lis-HCJ2. The washed bacterial cells were suspended in 5 ml of the above buffer solution and disrupted by ultrasonication at 0°C [Branson Sonic Power Campa =
(Branson 5onic PowerComp
any ) company, sonifier cell disruptor (5onifier cell disruptor)
) 200. output control
control) 2. Processed for 10 minutes]. This was centrifuged at 15,000 rpm for 30 minutes to obtain bacterial cell residue. From this bacterial cell residue, the method of Marston et al.
Nie O Marston (F.

A, OoMarston et al.: Biotec-hnology 2.800 (1
The chum salmon prolactin polypeptide derivative was extracted, purified, solubilized, and regenerated using the method (984)), and the growth promoting activity of the chum salmon prolactin polypeptide derivative was measured by the following method.

Measurement of chum salmon prolactin activity: Juvenile rainbow trout (average weight 12 g) were divided into groups of 10 and reared in a circulating tank at a water temperature of 15°C. The feed was a compounded raw material (first 4C, manufactured by Nippon Compound Feed Co., Ltd.), which was given at 4% of the body weight per day for the first 9 days, and then 3% in two divided doses. After dissolving the chum salmon brolactin polypeptide derivative in a small amount of 0.01N hydroxylated IJum aqueous solution,
% sodium chloride aqueous solution was added to give a concentration of 1.15%.

This was injected into the pus cavity at 1 μg per 1 g of body weight. The control group received only 0.9% sodium chloride aqueous solution. Injections were performed 5 times every 5 days and body weights were measured at the same time. As a result, plasmids of the present invention pSPRL (121! and p
It has been revealed that a prolactin polypeptide derivative encoded by SPRLQ52 promotes the growth of rainbow trout.

Reference Example I Construction of ATG vector pTrs20: According to the procedure shown in Fig. 2, ATG vector pTrs20 was constructed having a distance of 14 bases between the SD sequence and the ΔTG start codon and a Sac■ site immediately after the ΔTG codon. .

First, pKYPlo 3■ prepared by the method described in JP-A-58-110600 was dissolved in 30% of Y-100 Enoki's solution, and restriction enzyme Ban■ and restriction enzyme NruI (manufactured by Ny England Biolabs) were added at 60% each. Each unit was added and the cleavage reaction was carried out at 37°C for 3 hours. Approximately 3.8KM)D containing Ptrp was extracted from this reaction solution by the LGT method.
Approximately 0.5 μ of NA fragment (BanII [-NruI fragment) was obtained.

On the other hand, in order to add an ATC start codon downstream of Ptrp, the following DNA linker was added using the triester method CR,Cr
ea et al: Proceedings of the National
Academy of Science (Proc. Natl.
, Acad, Sci,) [ISA, 75.576
5 (1978)).

Synthetic NA of 19-+net and 17-mer (each 1
0 pmol each) to 5QmM) Lis-HCjl!
(p) 17.5).

10 m M VI g CR2, 5 m M dithiothreitol.

Dissolve in a total of 120 JIIl solution containing 0.1mM EDTA and 1mM ATP, add 3 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.), and incubate at 37°C for 6 hours.
The phosphorylation reaction was performed for 0 minutes.

Next, BanI[[-N
Dissolve 0.1 g of ruJ fragment (approximately 3.8 Kb) and approximately 0.5 picomole of the above DNA linker in T4 ligase buffer 204, add 2 units of T4 DNA IJ gauze, and conjugate at 4°C for 18 hours. The reaction was carried out.

Using the mixture of recombinant plasmids obtained, E. coli 8
8101 stocks [Bol 1ver et al. Niji-7
(Gene) 2.75 (1977)) to obtain Ap' colonies. Plasmid DNA was recovered from the cultured cells of this colony. 1. The structure of the plasmid was determined using the restriction enzymes EcoRI, Ban1I [, Hind
After cleaving with m, 5acl, and NruI, it was confirmed by agarose gel electrophoresis. This plasmid was named pTrs20 (Figure 2). Hi
It was confirmed using the Sanger method using M13 phage that the base sequence near the ndII site was as follows.

Effects of the Invention According to the present invention, a recombinant DNA incorporating a DNA encoding a prolactin polypeptide derivative of Shirojike
Microorganisms containing the recombinant DNA are obtained and can be used for mass production of chum salmon prolactin polypeptide derivatives.

[Brief explanation of drawings]

Figure 1 shows plasmids pSPRL028 and pSPR
It is a flow sheet showing the creation process of LQ52. FIG. 2 is a flow sheet showing the steps for constructing plasmid pTrs20. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. -! /

Claims (9)

[Claims] (1) A fish prolactin polypeptide derivative having the peptide sequences shown in Tables 1 and 2. (2) The polypeptide derivative according to claim 1, wherein the fish prolactin polypeptide derivative is a hormone having a growth promoting effect on herring (Clupeiformes). (3) DNA encoding a fish prolactin polypeptide derivative. (4) The DNA according to claim 3, wherein the fish prolactin polypeptide derivative has the peptide sequences shown in Tables 1 and 2. (5) A recombinant DNA incorporating DNA encoding a fish prolactin polypeptide derivative having the peptide sequences shown in Tables 1 and 2. (6) Plasmids pSPRLQ28 and pSPRLQ
Recombinant D according to claim 5 named as 52
N.A. (7) A microorganism containing a recombinant DNA incorporating DNA encoding a fish prolactin polypeptide derivative having the peptide sequences shown in Tables 1 and 2. (8) The microorganism according to claim 7, wherein the microorganism belongs to Escherichia coli. (9) A microorganism containing a recombinant DNA into which a DNA encoding a fish prolactin polypeptide derivative has been incorporated is cultured in a nutrient medium, a fish prolactin polypeptide derivative is accumulated in the culture, and a fish prolactin polypeptide derivative is extracted from the culture. A method for producing a fish prolactin polypeptide derivative, which comprises collecting the polypeptide derivative.