JPH01174387A - Caprine growth hormone - Google Patents

Abstract

PURPOSE:To enable mass production of a caprine growth hormone, by collecting a cDNA capable of coding a caprine growth hormone precursor, preparing a recombinant DNA containing the cDNA and forming a transformant. CONSTITUTION:An mRNA is extracted from a caprine pituitary gland and a cDNA capable of hybridizing a bovine growth hormone gene (cDNA) is selected using the cDNA as a probe to clone a cDNA capable of coding the caprine growth hormone. A DNA (cDNA) having complementation to the mRNA of the caprine growth hormone is prepared using the above-mentioned mRNA as a template and a recombinant plasmid integrating the afore-mentioned DNA is subsequently prepared. Thereby a microbial cell, such as Escherichia coli, can be transformed therewith and cultivated to collect the caprine growth hormone in a large amount.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to goat growth hormone, goat growth hormone precursor, DNA encoding goat growth hormone, CDNA encoding goat growth hormone precursor, cDNA
and E. coli into which the recombinant plasmid containing cDNA has been integrated.

[Prior Art] Mammalian growth hormone is secreted by the pituitary gland and has growth-promoting effects, protein anabolism-promoting effects, lipid metabolic effects,
It is a peptide hormone that exhibits physiological activities such as sugar metabolism and electrolyte metabolism.

Human growth hormone is used as a therapeutic agent to promote growth in pituitary dwarfism. On the other hand, animal growth hormone
It is attracting attention mainly in the livestock industry from the viewpoints of increasing feed efficiency, improving meat quality in meat animals, and increasing milk production in dairy animals.

Goats are raised as livestock in regions with harsh climates and features, and are mainly used for milk, meat, and goat hair, and are the most important livestock in some regions. Goats are also frequently used as laboratory animals in the production of vaccines and antisera. Therefore, in goats as well as in other livestock, it is desired to increase feed efficiency, improve meat quality, and increase milk production.

17= However, although the presence of growth hormone in goats was presumed, it had not been isolated until now, and of course the amino acid sequence had not been determined.

Conventionally, growth hormones have been isolated only from rabbits, cows, sheep, and some fish.

[Problems to be Solved by the Invention] Here, it is also conceivable to administer growth hormones from closely related cows, sheep, etc. that have already been isolated for the above purpose to goats. However, different animal species generally have different hormone structures, and it is known that administering hormones from different species can produce antibodies against the hormones, resulting in undesirable effects. The amino acid sequence of goat growth hormone revealed by the present inventor has the sequence as described later in this specification, and includes bovine and sheep growth hormone [M, 0. Dayhoff, “At1as o
f Protein 5 sequence and 5t
National Bioche
mical Foundation, Washing
ton D, C,, Vol, 5 (+972), 5u
ppl, + (+973), 5upp1.2 (+9
76)] differed by 1 to 3 amino acids.

Furthermore, the goat growth hormone obtained from the pituitary gland of a goat is very small and therefore very expensive. Therefore, goat growth hormone cannot be extracted from the goat pituitary gland and used for the purposes of increasing feed efficiency, improving meat quality, increasing milk production, etc. Therefore, an object of the present invention is to provide goat growth hormone in large quantities at low cost.

[Means for Solving the Problems] The present inventor conducted research on a method for producing goat growth hormone using recombinant DNA technology.

As a result, a cDNA encoding a goat growth hormone precursor that can be used for goat growth hormone production was collected, and a recombinant DNA and a recombinant DNA containing this cDNA were collected.
A transformant containing A was successfully constructed. That is, Metsenger RNA (mRNA) is extracted from the goat pituitary gland, and using the bovine growth hormone gene (cDNA) as a probe, a cDNA that hybridizes with this is selected to encode goat growth hormone. cD
We succeeded in turning NA into a loan. Furthermore, this c D
The nucleotide sequence of NA was determined and the present invention was completed.

There have been no reports on the amino acid sequence of goat growth hormone, the amino acid sequence of goat growth hormone precursor, and the base sequence of cDNA of goat growth hormone precursor, and this is the first time that they have been clarified. Therefore,
All DNA base sequences that can encode goat growth hormone without changing the amino acid sequence of goat growth hormone are included in the present invention.

Here, the goat growth hormone precursor refers to a protein that exists outside cells and has a secretion signal peptide added to its N-terminus. Normally, secretory proteins are synthesized intracellularly, and during secretion, the signal peptide is excised and the mature form becomes [Blo
bel, G., and Dobberstein, B.
, J, Ce1l Biol, 67, 835 (19
75)]. The goat growth hormone of the present invention encodes the entire amino acid sequence. Furthermore, the cDNA of the present invention encodes the entire amino acid sequence of goat growth hormone precursor. Therefore, goat growth hormone can be produced using the full length of this cDNA or using a cDNA from which the portion encoding the secretory signal peptide has been removed.

The present invention will be explained in detail below.

The present invention provides goat growth hormone having the following amino acid sequence.

(Ala)n Phe Pro Ala Met Se
r Leu Ser Gly Lea"Phe Ala
Asn Ala Vat Leu Arg Ala
Gln His2゜Leu His Gln Leu
Ala Ala Asp Thr-Phe Lys30
Glu Pbe Glu Arg Thr Tyr l
ie Pro Glu Gly'.

Gin Arg Tyr Ser lie Gln A
sn Thr G1l1Van"Ala Phe Cy
s Phe Ser Glu Thr lle Pro
Ala”Pro Tbr Gly Lys Asn
Glu Ala Gin Gln Lys70Ser
Asp Lea Glu Leu Leu Arg l
Ser Leu”Leu Leu lle 'G
In Ser Trp Leu Gly Pro Le
u90Gin Phe Leu Ser Arg Va
l Phe Thr Asn Ser"'Lea Va
l Pbe GIy Thr Ser Asp Arg
VaI Tyr”0Glu Lys Leu Lys
Asp Leu Glu Glu Gly lie】
2゜Leu Ala Leu Met Arg Gin
Leu Glu Asp Van13'Thr Pr
o Arg Ala Gly Gin lie Leu
Lys G1n140=11- Thr Tyr Asp Lys Phe Asp T
br Asn Met Arg”.

Ser Asp Asp Ala Leu Leu L
ys Aso Tyr Gay"'Leu Leu S
er Cys Phe Arg Lys Asp Le
u His”.

Lys Thr Glu Tbr Tyr Leu A
rg Val Met 1. ys"'Cys Arg
Arg Phe Gly Glu Ala Ser C
FS Ala'9°Pbe (in the above sequence, n is 0 or 1) The hormone can be produced using recombinant DNA techniques as described below. That is, using goat growth hormone mRNA as a template, a DNA (cDNA) complementary to the mRNA is extracted.
A) is prepared and a recombinant plasmid incorporating the cDNA is prepared. Furthermore, the recombinant plasmid can be used for the amplification of goat growth hormone DNA, especially in bacteria such as E. coli. Microorganisms containing the plasmid are useful for producing goat growth hormone in large quantities at low cost.

Therefore, the present invention provides DNA encoding goat growth hormone.
, 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 procedure.

Total RNA was prepared from goat pituitary gland, and this was mixed with oligo-dT.
Cellulose (oligo dT cellulose)
RNA having polyadenylic acid (polyA) (poly(A)'' RNA) is separated by passing it through a column.

Using this poly(A)'' RNA as a template, double-stranded DNA is synthesized using reverse transcriptase.The recombinant is synthesized using DNA recombination technology, and a vector D
It is obtained by inserting the synthetic NA into NA. A recombinant plasmid having DNA complementary to goat growth hormone mRNA is then selected.

Next, the method for producing the DNA and recombinant plasmid of the present invention will be specifically explained.

The pituitary gland is removed from the goat, and guanidine thiocyanate is added immediately to crush it and solubilize it. Next, it was layered on a cesium chloride solution layer, and after ultracentrifugation, the whole cytoplasm R was deposited as a precipitate.
Get NA.

The extracted RNA was dissolved in a highly concentrated solution of potassium chloride,
Adsorb onto oligo dT cellulose column. 10mM
The mRNA with polyA is isolated by elution using a low salt solution such as Tris-HCl buffer (pH 7.5).

Hereinafter, cDNA synthesis [O
Kayama H, and Berg P, Mo1
．． Ce11. l1iol.

L 161 (1982)] and its integration into a vector. Poly(A)'' RNA, vector primer DNA (e.g. pcDV-1) were dissolved in Tris-HCl buffer (e.g. 50mM, pIIll, 3), magnesium chloride (e.g. 8mM), potassium chloride (e.g.
0111M), dithiothreitol (e.g. ImM)
, dATPldTTP, dCTP, and dGTP (for example, 2+nM each), reverse transcriptase is allowed to act at a constant temperature (for example, 42° C.) for a certain period of time (for example, 1 hour). The thus obtained RNA-DNA duplex was mixed with Tris-HCl buffer (e.g. 30mM, pH 6,8), sodium cacodylate (e.g. 138mM), cobalt chloride (e.g. 1mM), dithiothreitol (e.g. 0.1
Terminal nucleotidyl transferase is applied in a solution containing poly(rA) (e.g. 0.5Bg#n) and dCTP (e.g. 0.07mM) at a constant temperature (e.g. 37°C) for a certain period of time (e.g. 3 minutes). hand,
Add 10 oligo da chains to the 3' end of the RNA-DNA duplex.
Add around 100 pieces. This DNA was added to Tris-HCl buffer (e.g. 1mM, pH 7.5), magnesium chloride (e.g. 7111M) and sodium chloride (e.g. 60M).
cleavage with Hindl11 in a solution containing (mM). Add linker DNA (e.g. pL-x) to this cut DNA.
and Tris-HCl buffer (e.g. 18mM, pH
7,5), magnesium chloride (e.g. 4mM), ammonium sulfate (e.g. 9mM), potassium chloride (e.g. 92mM), β-nicotinamide adenine dinucleotide (β-NAD) (e.g. 0.111M) and bovine serum albumin (BSA). ) (e.g. 0.05mg/mn)
in a solution containing E. coli DNA ligase at a constant temperature (
For example, at 12°C), the cells are incubated for a certain period of time (for example, 16 hours). In this way, cDNA and linker DNA are circularized. This reaction solution contains dATP, dTTP, d
Add GTP and dCTP to a final concentration of 40 μM and β-NAD to a final concentration of 125 μM, and add E. coli DNA.
A ligase, E. coli polymerase! , E. coli ribonuclease H is added to convert the RNA portion into DNA, thereby obtaining a recombinant plasmid containing a complete double-stranded DNA.

Using the thus obtained recombinant plasmid, Escherichia coli (e.g. E. coli DHI) was grown using the rubidium chloride method [Hanaha
n, D,, J, Mo1. Biol, 166.55? (
19g3)]. Since the recombinant plasmid obtained above contains an ampicillin resistance gene, the transformed E. coli exhibits ampicillin resistance. The following method is commonly used to select from these ampicillin-resistant (Ap') strains a strain that carries a novel recombinant plasmid DNA that has a gene complementary to goat growth hormone mRNA. be. That is, the transformed strain obtained above is immobilized on a nitrocellulose filter, hybridized with a known bovine growth hormone cDNA probe, and those that hybridize strongly are selected. Selection by DNA probe was performed using Southern [San1
hern, E. M., J. Mo1. Biol, 9B,
503 (1975)], and by this method, recombinant DNA having DNA complementary to goat growth hormone mRNA can be identified. A recombinant plasmid can be prepared by culturing E. coli containing the recombinant plasmid thus identified. From this plasmid, the base sequence of the DNA fragment encoding the goat growth hormone precursor was determined, and after translation into an amino acid sequence, the amino acid sequence of the growth hormone precursor of a closely related animal (for example, a cow) was determined [Woychik, R. P...
eL al,, Nucl, Ac1ds Res, I
O, 7197 (19112)], the cDNA obtained in the present invention was found to be the cD of goat growth hormone precursor.
It is possible to confirm that it is NA.

The novel recombinant plasmid of the present invention can be used for mass production of goat growth hormone by introducing it into microorganisms such as E. coli or eukaryotic cells.

[Example] Examples of the present invention are shown below.

Example 1 Preparation of poly(A)'' RNA from goat pituitary gland: Guanidine thiocyanate-cesium chloride method from goat pituitary gland [Chirgwin, J.M., eL al.
.

Biochemistry 1B, 5294 (197
9)], polyA-containing RNA was prepared as follows.

-Approximately 0.3g of the goat's pituitary gland is added to 6M guanidine.
Thiocyanate, 5mM sodium citrate (pH 7,
5) , 0.5% Sarcosyl NL97, and O,IMβ
- Solubilized by crushing with a homogenizer in a solution 1stQ consisting of merkabutethanol. The homogenate was passed through an 18G needle several times to cleave the DNA. 5.7
MC5CQ, 0. I M E D T A (pl,
2.5 mfi of each solution of 0) was dispensed into ultracentrifuge tubes, and the homogenate was layered thereon. 30. with Hitachi RPS40T rotor. OOOrpm, after centrifugation for 12 hours, RNA
was recovered as a precipitate. After washing the RNA precipitate with 95% ethanol and drying, add 400 μa of 10 mM Tris-HCl buffer (pH 8,0) containing 5 mM EDTA.
After removing protein with chloroform and butanol,
It was recovered by ethanol precipitation. The obtained RNA was dissolved in 0°BmM) Lis-HCl buffer containing 5M potassium chloride (pH
After dissolution in 7,5), the mixture was incubated at 70°C for 13 minutes and then rapidly cooled, and the mixture was applied to an oligo dT cellulose column. The adsorbed polyA-containing mRNA was eluted with 10mM Tris-HCl buffer (pH 7.5), and the polyA-containing mRNA
RNA was obtained.

Example 2 cDNA synthesis and insertion of the DNA into a vector cDNA was synthesized and a recombinant plasmid incorporating it was prepared according to the method of Okayama-Barb. An outline of the process is shown in FIG.

4 pg of poly(A)+RNA prepared above was incubated in 15 mM Tris-HCl buffer (pH 7,5) at 65°C for 3 minutes, and then rapidly cooled. RN A guar for this
30 units of d (manufactured by Pharmacia) and 1μ of vector primer (pc DV-1, manufactured by Pharmacia)
g, dNTP (dATP, dTTP, dGTP and d
CTP) 2mM each, Tris-HCl buffer (pH 8,3)
50mM of magnesium chloride, 8mM of potassium chloride, and 1mM of dithiothreitol, and 40 units of reverse transcriptase (manufactured by Seikagaku Corporation) were added and incubated at 42°C for - hours. RNA complementary to mRNA was synthesized. The reactant is treated with phenol.
After chloroform extraction and ethanol precipitation, RNA-D
Vector primer DNA with an added NA double strand was recovered.

The DNA was mixed with 1mM cobalt chloride, 140mM sodium cacodylate, 0.1mM dithiothreitol, 30mM
Tris-HCl buffer (pH 6,8), O, IM d C
Dissolved in 40 μa of a solution consisting of T P and 0.5 μg/μa polyA (manufactured by Pharmacia), added 30 units of terminal deoxynucleotidyl transferase (manufactured by Pharmacia), and incubated at 37° C. for 2 minutes. Polydc@ was added to the 3' end of the cDNA. The reaction product was extracted with phenol and chloroform, and cDNA-vector primer DNA with a dc strand added was recovered by ethanol precipitation. The DNA was dissolved in 10mM Tris-HCl buffer (pl+7.5), 7mM magnesium chloride,
It was dissolved in 50 μa of a solution consisting of 60 mM sodium chloride, 20 units of Hind2O-Ill (manufactured by Takara Shuzo Co., Ltd.) was added, incubated at 37° for 0.2 hours, and cleaved at the HindI11 site.

The reaction product was extracted with phenol/chloroform and precipitated with ethanol to obtain dc strand-added cDNA beta primer DNA.
I got an A. The DNA and 0.3 μg of linker DNA (
pL-1, manufactured by Pharmacia) was added to 80μa of a solution consisting of 8mM Tris-HCl buffer (pH 7.5), 0.8mM EDTA and 0.1M sodium chloride at 65°C for 5 minutes and at 42°C. After incubating for 1 hour, it was gradually cooled to room temperature. 20mM Tris-HCl buffer (pH 7,5), 4mM magnesium chloride, 1
0mM ammonium sulfate, 100mM potassium chloride, Q
, 1mM β-NAD, and 0.05mg bovine serum albumin (BSA), and a total volume of the reaction solution was prepared to be 800 μa. 5.3 μg of E. coli DNA ligase was added to the reaction solution, and the mixture was incubated at 12° C. overnight. The reaction solution was treated with 33 μM each of dNTP, 125
Additional components were prepared to give μM β-NAD, and 3.6
μg Escherichia coli DNA ligase (manufactured by Pharmacia) and 8
Unit of Escherichia coli liponuclease H (manufactured by Pharmacia)
and 14 units of Escherichia coli DNA polymerase I (manufactured by Pharmacia) were added, and the mixture was incubated at 12°C, 15°C, 20°C, and 25°C for 30 minutes each. In the above reaction, C
Circularization of recombinant DNA containing DNA and RNA-DNA
The double-stranded RNA portion was replaced with DNA, producing a complete double-stranded DNA recombinant plasmid.

Example 3 Selection of recombinant DNA containing goat growth hormone cDNA Using the recombinant plasmid obtained in Example 2, E. coli DH
I was transformed according to the rubidium chloride method of Hanahan et al. Approximately 20,000 colonies obtained were fixed on a nitrocellulose filter. Bovine growth hormone cDNA 32
Forty strains that strongly hybridized to the p-labeled globe at 68°C were selected. It was confirmed that the obtained recombinant plasmids of 40 strains hybridized with the above probe by Southern's method. Among the recombinant plasmids of this strain, the one with the longest inserted part was designated as pgcGH-24.
It was named.

Example 4 Base sequence of the plasmid pgcGH-24 Plasmid I) gcGH-24 obtained above was digested with various restriction enzymes, and a restriction enzyme cleavage map of the cDNA portion was determined. The restriction enzyme cleavage map is shown in FIG. In the figure, loco indicates a region encoding a structural gene, - indicates a regulatory region that is not translated into protein, and - indicates a vector portion.

Next, the entire nucleotide sequence of the transcribed region of the plasmid pgcGH-24 was determined by Sanger's dideoxy method using M13 phage [Sanger F, et xi, P
roc, Natl, Acad, Sci, USA? 4.
5463 (1977)]. The sequences are shown in Table 1.

Table 1 AGGATCCCAG”GACCCAGTTC”ACC
AGACGAC3'TCAGGGTCCT'°GCTG
ACAGCT'°CACCAACTATG ATG G
CT GCA GGCCC'CCGG ACCTCC"
MeL Met Ala Ala Gly Pro A
rg Tbr 5erCTG CTCCTG GC
T TTCACCCTG CTCTGC""Leu
Le++ Lea All Phe Tbr Leu
Lea CysCTG CCCTGG ACT CA
G GTG GTG GGCGCC'3'Leu Pr
o Trp Thr Gln Val Val Gly
A11TTCCCA GCCATG TCCTTG
TCCGGCCTG"'Phe Pro Ala Me
t Ser Leu Ser Gly LeaTTT
GCCAACGCT GTG CTCCGG GCT
CAG””Pbe Ala Asn Ala Val
Leu Arg Ala GinCACCTG CAT
CAA CTG GCT GCT GACACC”.

His Lea His Gln Lea Ala A
la Asp ThrTTCAAA GAG TTT
GAG CGCACCTACATC"'Pbe Lys
Glu Pbe Glu Arg Tbr Tyr
1ieCCG GAG GGA CAG AGA TA
CTCCATCCAG"'Pro Glu Gly G
ln Arg Tyr Ser Ile Gln-24
= AACACCCAG GTT GCCTTCTG
TTCTCT3°IAsn Thr Gln V
al Ala Phe Cys Phe 5
erGAA ACCATCCCGGCCCCCA
CG GGCAAG"'Glu Thr Ile
Pro Ala Pro Thr Gly
LysAAT GAG GCCCAG CA
GAAA TCA GACTTG"'Asn
Glu Ala Gin Lys
Ser Asp LeaGAG CTG C
TT CGCATCTCA CTG CTCCT
T””Glu Lea Leu Arg li
e Ser Leu Leu Leu ATCC
AG TCG TGG CTT GGG C
CCCTG CAG"'11e Gln Ser
Trp Leu Gly Pro Leu
GlnTTCCTCAGCAGAGTCTTC
ACCAAACAGC"'Phe Leu Ser
Arg Lal Phe Thr Asn
5erCTG GTG TTT GGCACC
TCG GACCGT GTC""Lea Va
t Pbe Gly Tbr Ser As
p Arg ValTAT GAG AAG
CTG AAG GACCTG GAG G
A.A.”

Tyr Glu Lys Leu Lys
Asp Leu Glu GluGGCATCC
TG GCCCTG ATG CGG GAG
CTG″17Gly Ile Leu Al
a Leu Met Arg Glu Le
aGAA GAT GTT ACCCCCCGG
GCT GGG CAGr'''CI++ A
sp Val Thr Pro Arg A
ha Gay GinATCCTCAG CAG
ACCTAT GACAAA TTT57'11e
Leu Lys Gin Thr Tyr
Asp Lys PheGACACA AAC
ATG CGCAGT GACGACGCG59'
Asp Thr Asn MeL Arg
Ser Asp Asp AlaCTG CT
CAAG AACTACGGT CTG CTC
TCC”5Leu Leu Lys Asn
Tyr Gly Leu Leu 5erTG
CTTCCGG AAG GACCTG CAC
AAG ACG652Cys Phe Arg
Lys Asp Leu His Lys
ThrGAG ACG TACCTG AGG
GTCATG AAG TGT"'Glu
Thr Tyr Leu Arg Val
Met Lys CysCGCCGCTTCGGG
GAG GCCA GCTGT GCC'°6A
r(Arg Phe Gly Glu Ala
Ser Cys AlaTTCTAG TTG
CCAGCCA722 TCTGTTGTTTA732
he CCCCTCCCCG"2 TGCCTTCCTA"2
GACCCTGGAA762GGTGCCACTC7
72CAGTGCCCAC782TGTCCTTTCC
792TAATAAAGCG802 AGGAAATT
GC”2 ATCA results for cattle and rats [Pag
e, G, S, et al.

Nucl, Ac1ds, Res, 9. 2087
(19ε1)], human [Denojo, P.
M,, et al,, Nucl, Ac1ds, Re
S, 9.3719 (1981)], the region encoding the goat growth hormone precursor and the start codon (methionine) were determined.
, the position and amino acid sequence of the stop codon were determined. As a result, it was revealed that the amino acid sequence of the goat growth hormone precursor obtained according to the present invention differs from that of the closely related cow by two amino acids. Furthermore, since the total number of amino acids matched that of bovine growth hormone precursor and the amino acid sequence had extremely high similarity, it was confirmed that the cDNA of the present invention is a cDNA of goat growth hormone precursor. Furthermore, by comparison with the amino acid sequence of adult bovine or sheep growth hormone, it was found that the N-terminal amino acid of adult goat growth hormone is the 27th amino acid from the N-terminus of the precursor, i.e., alanine, or the 28th amino acid, i.e., phenylalanine, from the N-terminus of the precursor. The polypeptide up to position 26 or 27 was considered to be a secretion signal peptide.

Incidentally, such N-terminal amino acid heterogeneity has also been reported for the amino acids of bovine growth hormone [Li, C,
)1. and Ash, L., J., BIol, ch.
em, 203.119 (1953)].

As described above, a polypeptide having goat growth hormone activity can be obtained by deleting the cDNA encoding the entire region of the goat growth hormone precursor of the present invention or the region encoding the amino acids 1 to 26 or 27 from the cDNA. It is possible to produce it using DNA obtained by

The E. coli containing pgcGH-24 was deposited as E. coli EGGH24 on December 9, 1985 at the Institute of Microbial Technology, Agency of Industrial Science and Technology (deposit number FERM P-9754).

[Brief explanation of the drawing]

Figure 1 shows an overview of cDNA synthesis by the Okayama-Barb method and the construction process of a recombinant plasmid containing the DNA, and Figure 2 shows a restriction enzyme cleavage map of the cDNA portion of pgcGH-24. . Primer + RNA

Claims (11)

[Claims] (1) The following amino acid sequence: [There is a gene sequence] (n in the above array is 0 or 1) Goat growth hormone expressed as (2) The following amino acid sequence: [There is a gene sequence] [There is a gene sequence] Goat growth hormone precursor expressed as . (3) DNA encoding goat growth hormone having the following amino acid sequence. [There is a gene sequence] (n in the above sequence is 0 or 1) (4) cDNA encoding goat growth hormone precursor
. (5) The following base sequence: [There is a gene sequence] The cDNA according to claim 4, which has the following. (6) cDNA encoding goat growth hormone precursor
Recombinant plasmid containing. (7) The recombinant plasmid according to claim 6, wherein the cDNA has the following base sequences: [There is a gene sequence] [There is a gene sequence]. (8) The recombinant plasmid according to claim 6, wherein the plasmid is pgcGH-24. (9) E. coli containing recombinant DNA into which DNA encoding a goat growth hormone precursor has been incorporated. (10) The E. coli according to claim 9, wherein the recombinant DNA is a plasmid. (11) The E. coli according to claim 9, wherein the plasmid is pgcGH-24. (12) The E. coli according to claim 9, wherein the E. coli is EGGH24.