JPH03219876A - Recombinant dna for producing rat prolactin and process for producing bacterial strain and rat prolactin - Google Patents

Abstract

PURPOSE:To enable the bacterial production of rat prolactin useful for the investigation of human prolactin by integrating a specific sequence and codon to the upstream of a gene coding rat prolactin polypeptide. CONSTITUTION:The objective recombinant DNA for the production of rat prolactin is produced by integrating a promoter, a Shine-Dalgarno sequence and an initiation codon in the order to the upstream of a gene coding a rat prolactin polypeptide having a molecular sequence, a part of which is shown in the figure. The above promoter is preferably a tac promoter. The Shine- Dalgarno sequence is a region supposed to play an important role generally in the start of the biosynthesis of proteins. The initiation codon is a genetic code to designate the start of protein synthesis. The genetic code is a segment composed of a sequence of three nucleic acid bases and consisting of three nucleotide bonds arranged in the order of ATG.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a DNA for rat prolactin production created by recombinant deoxyribonucleic acid (hereinafter referred to as DNA) technology. This invention also provides the rat prolactin production DNA thus produced.
This relates to a transformed bacterium obtained by introducing the above into a bacterium.

Furthermore, the present invention relates to a method for producing rat prolactin by culturing the bacteria thus obtained.

(Prior Art) Human prolactin is a peptide hormone with a molecular weight of 22,000 that is secreted from the pituitary gland.

Human prolactin is also called luteotropin, luteinizing hormone, and lactogenic hormone.

Human prolactin is presumed to have the effect of promoting mammary gland development, initiating and maintaining milk secretion, and promoting the growth of the prostate and seminal glands, but much remains unknown. The reason for this is said to be that only trace amounts of human prolactin can be obtained.

It is generally believed that mammalian prolactins have similar actions. Therefore, studying rat prolactin is of great help in studying human prolactin. However, since the amount of rat prolactin obtained is minute, it is difficult to investigate its properties, and its effects are not well understood. Therefore, there has been a demand for a method that can obtain more rat prolactin (hereinafter referred to as rPRL).

On the other hand, regarding rPRL gene cloning, E.
Nucleic, amide, by J. Gubbins et al.
Lisa-1-(Nucleic Ac1ds Res
6°915-93 (1 (1979):), and by N. E. Cooke et al. in the Journal of Biological Chemistry (J. Biol Ch.
Em,) 255°6502-6510 (1980). According to this, rPRL is said to have a nucleotide sequence as shown in Table 1.

(Problems to be Solved by the Invention) This invention solves the problem, as described above, even though rPRL is predicted to be a useful substance in carrying out research on human prolactin, the amount obtained is small. Focusing on the fact that neither actual efficacy nor physiological activity could be confirmed, the aim of this project is to make bacteria produce rPRL using recombinant DNA technology. Therefore, the first object of the present invention is to provide a recombinant DNA that, when introduced into bacteria, can produce rPRL inside the bacteria.

(Means for Solving the Problem) The inventor first developed a plasmid p that can be propagated in E. coli using an extract from the pituitary gland of a rat as a material.
rPRLl was synthesized. This plasmid pr
A new gene was created by inserting the promoter Shine Dalgarno sequence and a translation start codon in this order upstream of PRLl, and this was introduced into E. coli to create transformed E. coli. Subsequently, when this E. coli was cultured, it was confirmed that rPRL was produced there. This invention is
This was done based on such confirmation.

(Summary of the Invention) The first invention in this application is the rPR shown in Table 1.
This invention relates to a recombinant DNA for producing rPRL, which is made by incorporating a promoter, a Shine-Dalgarno sequence, and a translation initiation codon in this order upstream of the gene encoding L.

The second invention in this application is the recombinant DNA described above.
This relates to a transformed bacterial strain created by introducing this into bacteria.

The third invention in this application relates to a method for producing rPRL by culturing the above-mentioned transformed bacterial strain to produce rPRL.

(Obtaining rPRL Gene) The inventor first isolated Metsenger RNA having a polyA terminal from rat pituitary tissue. RNA having a polyA end means RNA having an adenine polymer chain at the 3' end. The inventor then determined the complementary DNA of this RNA according to the Okayamaberg method.
A was synthesized and incorporated into a vector plasmid, thus creating a plasmid prPRLl that contains rat prolactin complementary DNA and can be amplified in E. coli cells. Furthermore, it was confirmed that plasmid prPRL1 had the base sequence shown in Table 2.

Plasmid prPRLl shown in Table 2 contains the base sequence shown in Table 1 therein. Table 1 shows the base sequence of a gene in a single strand and places it in the upper row.
Table 2 follows the gene representation method of adding the corresponding amino acid in the lower row, but Table 2 follows the gene representation method of showing the base sequence as a double strand in two rows, the upper and lower rows, so Tables 1 and 2 Although it appears to be indicating something completely different, it is actually not. Counting the upper and lower rows in Table 2 as a set, "
AGTGG...'' base sequence completely matches the base sequence in Table 1. Therefore, plasmid prPRLl shown in Table 2 contains the base sequence shown in Table 1.

prPRLl shown in Table 2 is a cloned gene encoding rPRL and can only be propagated in E. coli. That is, even if prPRLl is introduced into E. coli, it cannot cause E. coli to produce rPRL. In this invention, a promoter, a Shine-Dalgarno sequence, and a translation start codon are incorporated in this order into prPRL1 to create recombinant DNA, and when this is introduced into bacteria, the bacteria can produce rPRL.

(Explanation of promoter) The promoter to be incorporated in this invention is as follows:
A promoter of the tac type is suitable. The tank-based promoter is deBoer, H.

A is Proc, Natl, Acad, Sci,
USA.

78, 21 (1983), and has the following base sequence.

(Explanation of Shine-Dalgarno array) Shine-Dalgarno array (hereinafter referred to as SD array) is
This region is generally thought to play an important role in the initiation of protein biosynthesis. The SD sequence is rich in purine bases and has a length of 3-9 bases.

(Explanation of the translation start codon) The translation start codon is a genetic code that specifies the start of protein synthesis. This genetic code consists of a sequence of three nucleobases, which consists of three nucleotide bonds arranged in the order of ATG.

The promoter, SD sequence, and translation initiation codon are all required to be integrated in sequence upstream of the gene encoding the rat rPRL polypeptide.

Furthermore, their integration sites must be at appropriate distances from the gene encoding the rPRL polypeptide and at appropriate distances from each other. In other words, the promoter is located from -10 to - from the start point of mRNA transcription.
35, and there are 6 to 18 base pairs between the SD sequence and the translation initiation codon.

If the SD sequence and translation initiation codon are not contained in the promoter-containing vector DNA to be incorporated, the SD sequence and translation initiation codon must be incorporated. However, if the SD sequence and translation start codon are
If the DNA sequence is already contained in a vector containing a desired promoter, there is no need to specifically incorporate the SD sequence and translation initiation codon.

Plasmid pKK223-3 can be used as the vector DNA containing the SD sequence and promoter. Plasmid pKK223-3 was obtained from deB.

er, H, A, Proc, Nat, Acad,
Sci, USA 78.21 (1983).

(Integration) In order to integrate the vector DNA containing the promoter etc. into the rPRL-encoding DNA, both DNAs are cut at appropriate positions using restriction enzymes, and then T4 DNA ligase is used to join them. This can be done by NA recombination techniques.

In this way, the DNA in which the promoter, SD sequence, and translation initiation codon are incorporated into the rPRL-encoding gene has, for example, the following base sequence.

Example of tac promoter (recombinant method) As an example of recombinant technology, plasmid prPRLl,
The outline of the method for creating plasmid pKK'RPIO from pKK223-3 will be explained below. First, an AccLRsal fragment encoding the C-terminal region of the mature rPRL polypeptide is obtained from plasmid prPRL1. Then, RsaI of this fragment (Hindll in aI)
1 linker.

On the other hand, create the D N A 'J linker shown below.

5'-AATTATGTTACCAGTTTGTT3'
-TACAATGGTCAAACAACAGGAGGA
GATTGTCAAACACGTCICTCCTCTA
ACAGTTTGTGCATTAC-3' (No, 1) GTAATGGC-5' (No, 2) 5'-CGGAGC! TGTTTGACCGTGTG3
'-CTCGACAAAAACTGGCACACGTCAT
GCT'l”TCTCAC!TACATOCAGTAC
GAAAGAGTGATGTAGCATACCCTGT
-3' (No, 3) GTATGGGACATA
-5' (No, 4) The cholera synthetic NA clinker contains a translation initiation codon followed by a DNA sequence that covers the vicinity of the N-terminus of mature rPRL.

Next, EcoRI-Hindl [[
Get the fragment.

Synthesis of the above two DNA fragments and two pairs N A IJ
Linkers 1 to 4 are ligated using T4 DNA ligase to obtain the recombinant plasmid pKKRPIO shown in FIG. This plasmid has a region encoding mature rPRL linked downstream of the taC bromotor.

As another example of recombinant technology, plasmid p KKRPI
Regarding the method of making plasmid pKcRP12 from O.
The outline is explained below. First, plasmid pK
A 5spI fragment containing the tac vector, SD sequence, and rPRL encoding region is obtained from KRP10.

Separately, plasmid pUC19F) L9Kbp Pv
Obtain u I[-8s p I fragment.

The above two DNA fragments are joined using T4DNA IJ gauze to obtain the recombinant plasmid pKCRP12 shown in FIG. This plasmid is a multicopy plasmid in which the plasmid replication origin is I) UCl3
It originates from.

(Recombinant reaction conditions) The reaction in the above recombinant technique can be carried out in the following manner.

Restriction enzymes and 'L'4 DNA described below! The J gauze made by Zenshuzo Co., Ltd. is used, and the definition of the unit of activity is according to the company's text CTakara Reagents f.

r Genetic Engineering R
e5earch (1987)).

DNA cleavage reaction with restriction enzymes is usually 0.1 to 20
py DNA at 2-200mM (preferably lO-40mM).
Tris(hydroxymethyl)aminomethane (mM)
HCI (hereinafter referred to as Tris-HCl) (pH 6.0 ~
9.5 preferably pH 7.0-8.0), O-200
NaCl of mAI, 2-30mM (preferably 5-1
Restriction enzyme 0 was added in a reaction solution containing 0 mM) Mg0+2.
．． The reaction is carried out using 1 to 100 units (preferably 1 to 5 units for lp9 DNA) at 20 to 70''C (optimum temperature for each restriction enzyme) for 1 to 24 hours.

The reaction can be stopped usually by heating at 55 to 70'C for 5 to 30 minutes, or by deactivating the restriction enzyme with a reagent such as phenol.

Extraction and purification of DNA fragments generated by restriction enzyme treatment can be performed by methods such as polyacrylamide electrophoresis and low melting point agarose methods.

The binding reaction of the DNA fragments is carried out using 2 to 200 mM (preferably 10 to 40 mM) Tris-HC1 (pH (3, l).
-9.5, preferably pH7.0-8.0), 2-2
0mM (preferably 5-10rnM) (7) Mg
C+2.0.1-10mM (preferably 0.5-2.
0 mM) of adenosine 5'-triphosphate (hereinafter referred to as ATP)
) 1 to 50 mM (preferably 5 to 10 mM
) in a reaction solution containing dithiothreitol, 'I'4D
Using 0.3-10 units of NA ligase at 1-37°C (
It is preferably carried out at 3 to 20'c) for 15 minutes to 72 hours (preferably 2 to 24 hours).

(Transformed bacteria) The recombinant plasmid DNA generated by the ligation reaction may be transformed using the transformation method of Cohen et al.
Cohen (S, N, Cohen) et al.: Proceedings of the National Academy of Sciences (Proc, Nat 1. Acad, F; ci)
), USA, 69, 2110 (1972)].

There is no need to use a particular strain of E. coli as a host for transformation. Among Escherichia coli, E, c
oIiDHI (ATCC33849), E, coliJ
M109. E,coIiHBlol(ATCC3369
4) is appropriate. Isolation of recombinant plasmid DNA from Escherichia coli carrying recombinant plasmid DNA was carried out by Birnboim (Bi
H.C. Birnboim et al.
Nucleic Ac1d Res
)7.1513 (1979)].

After cutting the plasmid DNA with 1 to 10 kinds of restriction enzymes,
Examine the cleavage site by agarose gel electrophoresis or polyacrylamide gel electrophoresis. Furthermore, if it is necessary to determine the base sequence of DNA, the Maxam-Gilbert method [Proceedings of the National Academia of Science (Proc, Natl., Ac.
ad, Sci.), 74° 560 (1977)) or the Sanger method using M13 phage [
Sanger et al.: Proceedings of the National Academy of Sciences (P.
roc, Natl, Acad.

Sci. USA, 74.5463 (1977)).

According to the present invention, rPRL polypeptides can be produced as follows. That is, the plasmid (
For example, E. coli is transformed using pKKRPlo, pKCRP12), and E. coli carrying each plasmid is selected from ampicillin-resistant colonies. The expression of the rPRL polypeptide in the transformed ampicillin-resistant E. coli can be confirmed by the colony immunoassay method described below. That is, a colony of a transformed strain grown on an agar medium is transferred to a nitrocellulose filter, the bacterial cells are lysed with lysozyme, and rPR
The presence of the L polypeptide was detected using an antibody against rPRL, protein A horseradish peroxidase conjugate (E-Y Laboratories) and 4-chloro-1
- Enzyme immunoassay method using naphthol (
P, Bucle and E, Zehelein: G
ene, 16.149 (1981)). By culturing Escherichia coli carrying these plasmids in an appropriate medium, rPRL polypeptide can be produced in the culture.

(Production of rPRL) The medium used here includes growth of E. coli and rPRL.
Both synthetic and natural media suitable for producing RL polypeptides can be used.

Carbon sources include glucose, fructose, lactose, glycerol, mannitol, sorbitol, etc.
As the nitrogen source, NH2O1, (NH4)2804, casamino acids, yeast extract, polypeptone, meat extract, batatotributone, corn stave liquor, etc. can be used.

Cultivation is carried out at pH 5.5 to 8.5 and temperature 18°C to 40°C by aerated agitation culture. Since rPRL accumulates in the cultured cells after 5 to 90 hours of culture, the cells are harvested from the culture, crushed, and centrifuged. From the resulting supernatant, the polypeptide is collected according to a conventional extraction method. The bacterial cells can be disrupted by treating the bacterial cells with lysozyme, followed by freezing and thawing, by ultrasonication, or by using osmotic pressure. In addition, the detection of the polypeptide can be performed by directly transferring the cultured bacterial cells using Laemmli's sample buffer [Laemmli, Nature,
227, 680 (1970):] and then subjected to 5DS-polyacrylamide gel electrophoresis [Rem! J (Laemm
The method of li) is carried out by Coomassie Brilliant Blue staining.

In addition, to confirm that the produced polypeptide is the same polypeptide as rPRL, use 5DS as described above.
- Polypeptides separated by polyacrylamide gel electrophoresis are electrically
C, W, N, Burnet: A
nal, Eiochem, 112, 195-203 (
1981)], and the prolactin band was detected using an antibody against rPRL and a protein A horseradish peroxidase conjugate (E
Enzyme immunoassay method CP using -Y Laboratories) and 4-chloro-1-+7toll, Buc
le and E, Zehelein: Gene.

16.149 (1981)].

(Example) Examples of the present invention are shown below.

Example 1 Expression of rPRL by tac promoter As a plasmid containing DNA encoding rPRL, 25% of the plasmid prPRLl shown in Table 2 was mixed with IOIIIM Tris-HCl (p18.0), 50IIIM N
It was dissolved in 300 μl of a solution containing aCl, 7 mM MgClz, and 7 mM 2-mercaptoethanol, 100 units of restriction enzyme Rsar was added, and digestion was performed at 37° C. for 2 hours.

This reaction solution was subjected to agarose gel electrophoresis, and then approximately 5 1.6 i DNA fragments were obtained by electroelution.

Next, 51Ig of this DNA and the 10mer shown below
Phosphorylated old ndlll linker DNA (manufactured by Takara Shuzo Co., Ltd.) 5'-pCCAAGCTTGG-3'3'-CGTTCGAACCp-5' 2 tails were treated with 66 mM Tris-HCI (pH 7,6), 6.
6mM MgCh, 10+H dithiothreitol, 1
T4D was dissolved in 20 pl of a solution containing mM (7) ATP.
Add 50 units of NA ligase (manufactured by Takara Shuzo Co., Ltd.) to 12.5
The binding reaction was carried out for 16 hours at °C. DNA was recovered from the reaction solution by ethanol precipitation. This DNA is 10
mM Tris-HCI (pH 7,5), 7mM Ngc
1. ．． Dissolved in 100Iffi of a solution containing 60mM NaCl (hereinafter referred to as old ndlll buffer),
100 units of the restriction enzyme old ndI was added and digestion was performed at 37°C for 2 hours.

DNA was recovered from the reaction solution by ethanol precipitation.

Next, the recovered DNA was added to 10 mM Tris-1 (cI).
pH 7,5), 7RIM MgCl, 60mM Na
C1,7d dissolved in 2-mercaptoethanol, 300 pR of a solution containing 0.01% bovine serum albumin,
50 units of restriction enzyme Accr were added and digestion was performed at 37°C for 2 hours. This reaction solution was subjected to polyacrylamide gel electrophoresis treatment, and then about two 522 bp Accl-HindT[l DNA fragments were obtained by electroelution.

Next, plasmid pKK223-3 (Pharmaci
(manufactured by company A) was dissolved in old ndl [[100% buffer solution,
Add 10 units of restriction enzyme) 1indnI and incubate at 37°C for 2
I did a time digest. DNA was recovered from this reaction solution by ethanol precipitation. This DNA was dissolved in 100 μl of EcoRr buffer, 10 units of restriction enzyme EcoRI was added, and digestion was performed at 37° C. for 2 hours. After subjecting this reaction solution to low melting point agarose gel electrophoresis, about 4 old ndll-EcoRI fragments of about 4.5b were obtained. - On the other hand, a DNA linker was synthesized for the purpose of adding a translation initiation codon ATG necessary for its expression to the DNA encoding mature rPRL, and for the purpose of ligating the vector DNA and the above DNA fragment. The synthesized DNA linker was made by the solid-phase phosphoramidite method, and the -terminal chain DN
A47mar (No. 1), 45mer (No. 2)
, 51mer (No, 3), and 51a+er (No,
There are four types (4), each with the following base sequence.

5'-AATTATGTTACCAGTTTGTTCA
GGAGGAGATTGTCA8＾CACCATTAC
-3' (inhibition 1) 3'-TACAATGGTCAA to CAAGTCCTC
CTCTAACAGTTTGTGGTAATGGC-5
'(Nα2) 5'-CGGAGCTGTTTGACCGTGTGGT
CATGCTTTCTCACTACATCCATACC
CTGT-3'(Nα3) 3'-CTCGACAAAACTGGCACACCAGT
ACGAAAGAGTGATGTAGGTATGGGGA
CATA-5' (Nα4) Among these, in order to phosphorylate the 5'-ends of Nα2 and Nα3, the following treatment was performed.

l nm of each single-stranded DNA of Nα2 and Nα3
ol in 50mM Tris-HCI (pH 9,5), 10
mM Mgcl, 51 dithiothreitol, 50n
In addition to solution 11d containing mol of ATP, 100% of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) was added to this mixture.
Unit was added and reacted at 37°C for 30 minutes to phosphorylate the 5'-terminus. After the reaction was completed, linker DNA was collected by ethanol precipitation.

100,100p of single-stranded DNA of kl and N[12 were each taken and dissolved in 10pβ of a solution containing 10mM Tris-HCI (pH 7.5) and 1mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA). Also, No,
3, No., and 4 single-stranded DNA each at 100pm.
o! and diluted with 101 Tris-HCI (pH 7.5).
) and EDTA of IIIM! dissolved in. Each synthetic DNA obtained in this way is
It was called a linker solution.

The Acc r-old ndll DNA fragment (522 bp) derived from the plasmid prPRLl obtained above and the plasmid pK
Hind m -EcoRI fragment derived from K223-3 (
0.028 pmol of about 4.5 Kb), 66 mM Tris-11cI (pH 7.6), and 6.61 MgC
1, and l(1+M dithiothreitol, III
M was dissolved in 20 ttl of a solution containing ATP, and 1 μl of each of the above synthetic DNA linker solutions was added thereto. 50 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added to this mixed solution, and a binding reaction was performed at 12,5''C for 16 hours.

This reaction solution was used to transform E. coli JM109 to obtain an ampicillin-resistant transformant. Among these strains, strains that produce rPRL were treated with antiserum against rPRL and protein A/horseradish peroxidase conjugate (manufactured by E-Y Laboratories).
and 4-chloro-1-naphthol by enzyme immunoassay method. Plasmid DNA was recovered from the rPRL producing strain to obtain plasmid pKKRP10 shown in FIG.

Plasmid pKKI? The structure of P10 is derived from the restriction enzyme Eco
It was cut with RI, Accl, and old ndI[l, and confirmed by agarose electrophoresis.

Example 2 Modification of replication origin 5t1g of plasmid pKKRP10 was mixed with 10mM Tris-HCI (pH 7,5) and 7IIIM-gcl□
was dissolved in 300μ2 of a solution containing 100mM NaCl and 7mM 2-mercaptoethanol, 20 units of restriction enzyme SsρI was added, and the mixture was reacted at 37°C for 2 hours. DNA was recovered from this reaction solution by ethanol precipitation. This DNA'A was mixed with 10mM Tris-MCI (pi
(8,0) and 0.1 wM EDTA, and bacterial alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.) at 0.1 wM.
4 units were added and reacted at 65°C for 2 hours to remove the 5'-position phosphoric acid group.

Next, 5μ of plasmid pU019 was mixed with 10+eM of Tris-1 (CI (pl(7,5)) and 71 of MgC1,
was dissolved in 300d of a solution containing 60+nM NaCI and 7mM 2-mercaptoethanol, and the restriction enzyme P
20 units of vu II and 20 units of Sspl were added and reacted at 37°C for 2 hours. After subjecting this reaction solution to agarose electrophoresis treatment, 1.9
Approximately one DNA fragment of Kbp was isolated.

Plasmid p K K It P obtained in Example 1, 1
0.028 pmol of the Ssp I fragment from plasmid pUC19 and 0.028 pmol of the Pvu U Ssp I fragment from plasmid pUC19 were added to 66 mM Tris-H
CI (pH 7,6), 6.6mM 1CI□, and 10
-20μ of a solution containing dithiothreitol and 1mM ATP! 50 units of T4 DNA ligase was added, and the binding reaction was carried out at 12.5°C for 16 hours.

This reaction solution was used to transform E. coli JM109 to obtain an ampicillin-resistant transformant. Among these strains, strains that produce rPRL were treated with antiserum against rPRL and protein A/horseradish peroxidase conjugate (manufactured by E-Y Laboratories).
And, it was selected by enzyme immunoassay method using 4-chloro-1-naphthol.

Plasmid DNA was recovered from the rPRL producing strain to obtain plasmid pKcIIP12 shown in FIG. The structure of plasmid pKcRP12 is based on the restriction enzyme old ndI [, Ac
cl, 5spI, and confirmed by agarose electrophoresis and polyacrylamide electrophoresis.

Example 3 In this example, rPRL was produced by E. coli harboring plasmid pKKRP10. The details are as follows.

E carrying the plasmid pKKRP10 obtained in Example 1
, coli JM109 was infected with 5-L-Broth (
Trypton of 1,0χ.

0.5! Yeast extract, 1.0x NaCl, 50!1 g/d ampicillin),
Cultured at 37°C, the growth of bacterial cells reached OD666nm = o
, 2 to 0.3, β-D-isopropylthiogalactoside (IPTG), an inducer of the tac promoter, was added to a concentration of 21 and incubated at 37°.
Culture was continued for 15 hours at C. The obtained culture solution was heated to 120
The cells were collected by centrifugation at 0 rpm for 10 minutes. The cells were suspended in Lae11111 sample bread, heated at 100°C for 10 minutes, and then suspended in Lae11111 sample bread.
mli method (Il, K, Laemmli; N
ature, 227.680-685 (1970)
5DS-polyacrylamide electrophoresis was performed according to Burnette's method (W, Neat Bu
rnette: Anal, Biochem,, 1
12.195-203 (1981)) were electroblotted onto nitrocellulose filters. Next, this nitrocellulose filter was treated with an enzyme immunoassay method using an antibody against rPRL and a protein A/horseradish peroxidase conjugate, and a prolactin band was detected at a molecular weight of 22,000. did. This hand could not be detected in E. coli that does not carry the plasmid. As a result, plasmid pK
It was found that E. coli harboring KRP10 produces rPRL.

E. coli harboring plasmid pKKRP10 was developed in 1999.
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FER P-11060 on October 20, 2015.

Example 4 In this example, rPRL was produced by culturing JM109 containing plasmid pKcRP12. The details are as follows.

E carrying the plasmid pKcRP12 obtained in Example 2
, coli JM109 was inoculated into the same L-broth as used in Example 3, and treated in exactly the same manner as in Example 3 to collect the bacterial cells. Furthermore, this bacterial cell was treated in exactly the same manner as in Example 3, and production of rPRL was confirmed.

Plasmid ρKCI? E. coli harboring P12 was identified as FERM P-11061 on October 20, 1999.
It has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(Effects of the Invention) According to the present invention, a recombinant DNA that expresses a DNA encoding an rPRL polypeptide in a microorganism can be obtained, and a fungus that retains this recombinant DNA can be obtained, thereby producing a large amount of rPRL. can be produced. If rPRL is produced in large quantities, it will be useful for elucidating the physiological activity of rPRL, and it will be useful for research on rats as industrial animals. Furthermore, if the physiological activity of rPRL is elucidated, it will contribute greatly to the elucidation of the physiological activity of human prolactin since its action is similar to that of mammalian prolactin.

(Brief explanation of the drawings) Figure 1 is a restriction map of plasmid prPRLl containing DNA complementary to lantoprolactin. Figure 2 is a diagram showing the construction process of plasmid pKKllP10. Figure 3 is a diagram showing the construction process of plasmid pKKllP10. It is a diagram showing the construction process of plasmid pKcIIP12.

Table 1 ACCGTGCTGT CCAAAGGGCT GAC
CTTTCGCCCGTCACTCGCGTTGCG
TTAAsnAsnCys*** CTTCAAAGGTTCTATTTGCATTACA
ACTTTCAGCACATGCTTAAGTATAA
TTGGTCTCTTTCTTAAAATAATAAAAAAA
CAAACTTTTAAAAATGρr? R Otsu1 (2) ρrPRL1 (3) ACAAGAATTT TCAGATAAAAG AAG
TTTCCAA GATAAAC(i-1-A AT(
j-1-1oAAA(aTTCCTCTTTT ATC
GCGTAGT CCGCGAGAAG GCGAAG
GAGCGAGTGACl(jAprPRLl(4) prPRLl(5) ACGATGTCTCAAGAACTTCA CCA
CCGGA-1-'l' (jAltauLuAIl
, J 1uAit-11ut-1GTAGGTCAGA
TAAτTAACAA CGGCCCTTCG
AT, CTCATTCA TCAA (jUl, jl
jLl;prPRLl(6) prPRLl(7) TATCCGCATA GTGCTCCGGG A
AA (jLPl, JAAu liLiAHindIn Figure 1, 3P revision

Claims (1)

[Claims] 1. A recombinant product for producing rat prolactin, which is created by incorporating a promoter, Shine-Dalgarno sequence, and translation initiation codon in this order upstream of the gene encoding the rat prolactin polypeptide shown in Table 1. DNA. 2. The gene encoding rat prolactin polypeptide shown in Table 1 was obtained from plasmid prPRLl, and the promoter, Shine-Dalgarno sequence, and translation initiation codon were combined with plasmid pKK233-3 and synthetic DNA.
linker, and these are ligated to form the plasmid p
The recombinant DNA for producing rat prolactin as set forth in claim 1, which is designated as KKR10. 3. The gene encoding the rat prolactin polypeptide shown in Table 1, the promoter, the Shine-Dalgarno sequence, and the translation initiation codon are contained in the plasmid pKKRP10.
and ligated with plasmid pUC19 to form plasmid pKCRP12, the recombinant DNA for producing rat prolactin according to claim 2
A. 4. A recombinant DNA for rat prolactin production created by incorporating a promoter, Shine-Dalgarno sequence, and translation initiation codon in this order upstream of the gene encoding the rat prolactin polypeptide shown in Table 1.
A bacterial strain for producing rat prolactin created by introducing this into bacteria. 5. The strain according to claim 4, wherein the bacterium is Escherichia coli. 6. Create a recombinant DNA for rat prolactin production by incorporating a promoter, Shine-Dalgarno sequence, and translation initiation codon in this order upstream of the gene encoding the rat prolactin polypeptide shown in Table 1;
A method for producing rat prolactin, which comprises introducing this recombinant DNA for producing rat prolactin into a bacterium to create a transformed bacterial strain, culturing the bacterium to produce rat prolactin polypeptide, and collecting the same.