CA2108834A1 - Isolated silver fox growth hormone gene, and a recombinant dna thereof - Google Patents

Abstract

ABSTRACT
The present invention provides a silver fox growth hormone gene encoding a polypeptide composes of a specific amino acid sequence, a silver fox growth hormone gene composed of a specific base sequence and a recombinant DNA comprising the silver fox growth hormone gene inserted into a vector DNA. The present invention is industrially extremely useful because silver fox growth hormone can be efficiently produced in a very short time by culturing in a medium an organism (e.g., a microorganism) carrying said recombinant DNA.

Description

~ 72813-38 ISOLATED SILVER FOX GROWTH HORMONE GENE, AND A RECOMBINANT DNA THEREOF
FIELD OF THE INVENTION
The present invention relates to an isolated gene encoding silver fox growth hormone and a recombinant DNA thereof.
BACKGROUND OF THE INVENTION
So far, no structure has been known for the growth hormone gene derived from a silver fox belonging to the super-family Canoidea and even the gene has never been isolated. It is known that animal growth hormone is a growth-promoting protein which is present in the animal pituitary and a purified growth hormone exhibits the growth promotion of a relevant animal when administered.
SUMMARY OF THE INVENTION
A first aspect of the present invention provides an isolated silver fox growth hormone gene encoding the polypeptide set forth in SEQ ID No. 2~ The isolated silver fox growth hormone gene is preferably that set forth in SEQ ID No. 1.
Another aspect of the present invention provides a recombinant DNA comprising the silver fox growth hormone gene inserted into -~
a vector DNA.
BRIEF DESCRIPTION OF THE DRAWING
FIG. l is a restriction map of recombinant plasmid pFGH300.
DETAILED DESCRIPTION OF THE INVENTION
An object of the present invention is to provide a silver fox growth hormone gene for producing silver fox growth 2 LO~$34 72813-38 hormone by the techniques of genetic engineering.
As a result of extensive research on silver fox-derived growth hormone, the present inventors have succeeded in isolation of the growth hormone gene and subsequent determination of its structure, to complete the present invention.
Hereinafter, the present invention is described in detail.
The gene donor used in the invention includes e.g. the pituitary derived from a silver fox belonging to the super-family Canoidea. .
From the silver fox pituitary tissue, m-RNA is prepared ~ ~ ~3~3~
dccording to a method as described e.g. in Proc. Natl. Acad. Sci.
U.S.A., vol. 70, p. 3646 (1973) or "Lab Manual Idenshi Kogaku"
(Laboratory Manual for Genetic Engineering), M. Muramatsu, page 70 (1988).
From the resulting m-RNA, cDNA can be synthesized according to a method as described e.g. in Mol. Cell. Biol., vol. 2, p. 161 (1982) or Gene, vol. 25, p. 263 (1983).
A wide variety of recombinant phage DNAs are obtained by inserting the above cDNA into a vector DNA, e.g. phage ~gtlO
(manufactured by Amersham). Then, ln vitro packaging of the recombinant DNAs into phage particles is carried out using cDNA
cloning system ~gtlO (manufactured by Amersham) by the adapter method instructed by the manufacture, and the phage particles were then transformed into organisms such as E. coli L87 (ob-tained from Amersham).
A cDNA coding for silver fox growth hormone (hereinafter referred to as "growth hormone cDNA") is obtained from the cDNA
library (vector: ~gtlO) by plaque hybridization with a mink growth hormone cDNA as a probe (see B.B.~.C., 173, No. 3, pp.
1200-1204 (1990)) on a Hybond blotting membrane (manufactured by Amersham) according to the manufacture's instruction.
The recombinant phage DNA thus obtained is treated with a restriction enzyme such as BamHI at 30-40 C, preferably 37 C, for 1-24 hours, preferably 2 hours, and then the reaction solution is subjected to agarose gel electrophoresis (Molecular Cloning, p.
150, Cold Spring Harbor Laboratory (1982)), whereby a DNA
containing a gene encoding the growth hormone derived from a silver fox (hereinafter referred to as "growth hormone gene") is obtained.
In the invention, any vector DNA can be used which includes plasmid vectorDNA, bacteriophagevectorDNA, etc. Specifically, DNAs such as plasmid pUC118, pUC119 and pBLUESCRIPTII (manufac-tured by Stratagene) are preferable. A cleaved vector DNA is obtained by digesting the above vector DNA with a restriction enzyme such as BamHI etc. (manufactured by Takara Shuzo Co., Ltd.).
The cleaved vector DNA is then mixed with the growth hormone gene-containing DNA prepared above, followed by treatment e.g.
with T4DNA ligase (manufactured by Boehringer Mannheim GmbH), thus giving rise to a recombinant DNA.
The recombinant DNA is transformed into e.g. E. coli K-12, preferably E. coli JM109 (obtained from Takara Shuzo Co., Ltd.), XLl-Blue (obtained from Funakoshi K.K.), etc., to give a transfo-rmant originating in each bacterial strain. Transformation can be effected according to the method described by D. M. Morrison (Methods in Enzymology, vol. 68, pp. 326-331 (1979)).
The growth hormone gene is determined for its base sequence according to the experiment under item (6) below. The base : ~ :
sequence thus determined is set forth in SEQ ID No. 1, and the amino acid sequence encoded by this base sequence is set forth in SEQ ID No. 2.

The present invention provides a silver fox growth hormone gene and a recombinant DNA containing the same, and is industri-ally extremely useful because the silver fox growth hormone can be efficiently produced in a very short time by culturing an ~ .
organism (e.g., a microorganism) carrying said recombinant DNA.

EXAMPLES
Hereinafter, the present invention is further illustrated indetail with reference to the Examples which, however, are not intended to limit the scope of the present invention. ~-Cloning of silver fox growth hormone cDNA ~
(1) Preparation of pituitary tissue ;
30 silver foxes (obtained from Zao Mink Ranch) were sacri-ficed and the pituitary was removed immediately. 0.6 g of -~
pituitary tissue was obtained. It was immediately immersed and frozen in liquid nitrogen, and then stored in a deep-freezer at 80 C (manufactured by Ebara Co., Ltd.).

(2) Preparation of RNA

50 mg of the pituitary obtained in item (1) above was treated according to the method described in Molecular Cloning, pp.

,. . ..

p ~ ,3 719-722, Cold Spring Harbor Laboratory(1989), to yield 150 ~g of RNAs in total.

(3) Synthesis of cDNA
Synthesis of cDNA was carried out using a kit manufactured by Boehringer Mannheim GmbH. According to the manufacture's instruction, 60 ~g of the above RNA was treated in accordance with the method described in Gene, vol. 25, p. 263 (1983), whereby 5 ~g of double-stranded cDNA was obtained.

(4) Preparation of cDNA library Using a kit manufactured by Amersham (cDNA cloning system ~gtlO, adapter method), 1 ~g of the above cDNA was subjected to the method indicated by the manufacture, thus giving rise to a cDNA library (about 2,000,000 strains).

(5) Screening of growth hormone cDNA fragments For screening of growth hormone cDNA, plaque hybridization was carried out on a Hybond blotting membrane (manufactured by Amersham) according to the manufacture's instruction. The details of the screening are as follows:
The probe used in plaque hybridization was prepared using a mink cDNA by Random Rrimer Extension Labeling System, NEP-103, manufactured by DuPont.
For screening of the silver fox growth hormone cDNA, plaque "- . : ., :
j;" ~

~88~ 728l3-38 hybridization was carried out using the above prove on a Hybond blotting membrane (manufactured by Amersham) according to the manufacture's instruction. One positive clone strain was obtained as a result of the screening of 10,000 strains from the cDNA library obtained under item (4) above.

., , (6) Analysis of the silver fox growth hormone gene From the isolated positive clone, a phage DNA was obtained according to a conventional method (Molecular Cloning, ed.
t Maniatis et al., pp. 77-85, Cold Spring Harbor Laboratory, USA, 1982), then cleaved with restriction enzymes such as BamHI, ~' EcoRI, etc., and analyzed by the aforementioned electrophoresis.
As a result, the silver fox growth hormone gene was estimated to i be present on a ca. 800 bp BamHI-cleaved DNA fragment. This fragment was subjected to agarose electrophoresis and purified with GENECLEAN II (purchased from Funakoshi K.K.). The resulting fragment was inserted into BamHI-cleaved pUCll9, thereby giving recombinant plasmid pFGH300 (see FIG. 1). According to a conventional method, this recombinant plasmid was transformed into XL1-~lue, to give a transformant E. col1 XLl-Blue(pFGH300).
This transformant, E. coli XL1-Blue(pFGH300), has beendeposited t with the accession No. FERM BP-4063 with Fermentation Research Institute, Agency of Industrial Science and Technology, Japan.
The base sequence of the obtained plasmid was determlned with a deletion kit for Kilo-Sequence (purchased from Takara Shuzo Co., Ltd.) using a 370 DNA Sequencing System (purchased from Applied siOSystems Inc.). The determined base sequence and the amino acid sequence encoded by the base sequence are set forth in SEQ ID
Nos. 1 and 2, respectively. The silver fox growth hormone gene possesses a coding region for 648 bases encoding 216 amino acids.

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~ ,EQUENCE LISTING
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SEQ ID NO~
~, SEQUENCE LENGTH: 648 ~;~
~ SEQUENCE TYPE: nucleic acid --~ ~ .
... .
, STRANDEDNESS: double ~ .
,i~ TOPOLOGY: linear ,~
:~7 MOLECULAR TYPE: mRNA to cDNA
... .
'-'1 ' ORIGINAL SOURCE
i'i, ORGANISM: silver fox (Vulpes vulpes) -, ~ SEQUENCE:
:,j ~.,i ,i~ ATG GCT GCA AGC CCT CGG AAC TCT GTG CTC CTG GCC TTC GCC TTG 4S

TAC ATC CCC GAG GGA CAG AGG TAC TCC ATC CAG AAC GCG ChG GCC 225 GhG GCC CAG CAG CGA TCC GAC GTG GAG CTG CTC CGC TTC TCC CTG 315 i~ CTG CTC ATC CAG TCG TGG CTC GGG CCC GTG CAG TTT CTC AGC AGG 360 AAG GCC GAG ACG TAC CTG CGG GTC ATG AhG TGT CGC CGC TTC GTG 630 GAA AGC AGC TGT GCC TTC

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~EQ ID NO: 2 SEQUENCE LENGTH: 216 SEQUENCE TYPE: amino acid TOPOLOGY: not known MOLECULAR TYPE: protein ORIGINAL SOURCE
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ORGANISM: silver fox (Vulpes vulpes) SEQUENCE:

Met Ala Ala Ser Pro Arg Asn Ser Val leu Leu Ala Phe Ala Leu Leu Cys Leu Pro Trp Pro Gln Glu Val l,ly Ala Phe Pro Ala Met ~5 30 ., .
Pro Leu Ser Ser Leu Phe Ala Asn Ala Val Leu Arg Ala Gln His I() 45 Leu His Gln Leu Ala Ala Asp Thr TYr l~s Glu Phe Glu Arg Ala S0 .)5 60 Tyr lle Pro Glu Gly Gln Ar~ Tyr Sel lle Gln Asn Ala Gln Ala , 0 75 Ala Phe CYS Phe Ser Glu Thr lle Pro Ala l'ro Thr GIY Lys Asp ~35 90 Glu Ala Gln Gln Arg Ser Asp Ual Clu leu Leu Arg Phe Ser Leu ., , !~ ' ~x~
; 5~ 100 10~
.Leu Leu lle Gln Ser Trp Leu GIY Pro Val Gln Phe Leu Ser Arg Val Phe Thr Asn Ser Leu Val Phe GiY Thr Ser Asp Arg Val TYr ` 125 130 135 Glu Lys Leu Lys Asp Leu Glu Glu G]y lle Gln Ala Leu Met Arg 1~0 14~ 150 Glu Leu Glu Asp Gly Ser Pro Arg Ala Gly Gln lle Leu LYS Gln .j ~
,; 155 160 165 Thr Tyr Asp LYS Phe Asp Thr Asn Leu Arg Ser ASP Asp Ala Leu ... .
~;1 170 175 180 Leu Lys Asn Tyr Gly Leu Leu Ser Cys Phe LYS Lys Asp Leu His Lys Ala Glu Thr TYr Leu Arg Val Met LYS CYS Arg Arg Phe Val G I u Ser Ser Cys A I a Phe ~, , ...
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Claims (8)

1. An isolated silver fox growth hormone gene encoding the polypeptide set forth in SEQ ID No. 2.

2. An isolated silver fox growth hormone gene set forth in SEQ ID No. 1.

3. A recombinant DNA comprising the silver fox growth hormone gene as defined in claim 1 or 2 inserted into a vector DNA.

4. The recombinant DNA according to claim 3, which is recombinant plasmid pFGH300.

5. The recombinant DNA according to claim 3, which is put in a bacteria by transformation.

6. The recombinant DNA according to claim 5, wherein the bacteria is E. coli.

7. The recombinant DNA according to claim 6, wherein the bacteria is E. coli. XL1-Blue.

8. Transformant E. coli. XL1-Blue (pFGH300).