JPH06153956A - Silver fox growth hormone gene and its recombinant dna - Google Patents

Abstract

PURPOSE:To provide a new gene suitable for the production of a growth hormone useful for promoting the growth of silver fox. CONSTITUTION:A silver fox growth hormone gene coding the polypeptide of formula. A c-DNA is synthesized from an m-RNA obtained from the pituitary gland of a silver fox of the family Canidae and integrated into phage lambdagt10. A gene bank library of cDNA is prepared by using the phage lambdagt10 as a vector. The objective growth hormone cDNA is selected and separated from the library by plaque hybridization selection using a mink growth hormone c DNA.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a silver fox growth hormone gene and its recombinant DNA.

[0002]

2. Description of the Related Art Conventionally, the structure of a growth hormone gene derived from a silver fox belonging to the family Canidae is completely unknown, and the fact is that the gene has not even been isolated. Animal growth hormone is a protein hormone that exists in the pituitary gland of animals and promotes growth. It is known that administration of purified growth hormone to the original animal has a growth promoting action.

[0003]

DISCLOSURE OF THE INVENTION The present invention aims to produce a silver fox growth hormone by a genetic engineering method, and to provide a silver fox growth hormone gene which is a source of the production.

[0004]

Means for Solving the Problems As a result of extensive studies on the growth hormone derived from silver fox, the present inventors succeeded in isolating this growth hormone gene and determining its structure, and completed the present invention. . That is, the present invention resides in the silver fox growth hormone gene encoding the polypeptide represented by SEQ ID NO: 2.

Further, the present invention resides in the silver fox growth hormone gene represented by SEQ ID NO: 1. Further, the present invention resides in recombinant DNA in which the silver fox growth hormone gene is inserted into vector DNA. Hereinafter, the present invention will be described in detail.

[0006] First, as a donor of the gene of the present invention, for example, a pituitary gland derived from a canine silver fox can be mentioned. Preparation of m-RNA from the above-mentioned Silver Fox pituitary tissue is carried out, for example, by Pronas (Proc. Natl. Acad. Sci. USA), 70, 3646 (197).
3), Laboratory Manual Genetic Engineering, Masami Muramatsu, p. 70 (198
8) It is performed by the method etc. described.

To synthesize c-DNA from the obtained m-RNA, for example, Mol.Cell.Bio is used.
l.), Volume 2, p. 161, (1982) and Gene, 25.
Volume, page 263 (1983). Then, the c-DNA thus obtained is used as a vector DNA, for example, phage λgt10 (manufactured by Amersham).
Etc. to obtain various recombinant phage DNAs. Using this recombinant phage DNA, for example, c-
DNA cloning system λgt10, in vitro packaging and E. coli L87 (obtained from Amersham) by the method described in the adapter method (Amersham)
Etc. can be transduced.

The detection of c-DNA encoding silver fox growth hormone (hereinafter referred to as growth hormone c-DNA) from the above various transduced strains is carried out as follows. From a gene bank library of c-DNA prepared using λgt10 as a vector, mink growth hormone c-DNA (BBRC, 173, No.3,1200 to 1204 (1
990)) for plaque hybridization.
Selection (Hybond blotting membrane protocol manufactured by Amersham, Inc.) was performed, and growth hormone c-
It can be obtained by selecting and separating DNA.

In order to obtain a DNA containing a gene encoding a growth hormone derived from silver fox (hereinafter referred to as a growth hormone gene) from the thus obtained recombinant phage DNA, the phage DN is used.
A restriction enzyme such as BamHI is allowed to act on A at a temperature of 30 to 40 ° C., preferably 37 ° C. for 1 to 24 hours, preferably 2 hours, and the resulting reaction solution is subjected to agarose gel electrophoresis (Molecular Cloning). , Page 150,
Cold Spring Harbor Laboratory (Cold
Spring Habor Laboratory) (1982)] containing a growth hormone gene derived from silver fox
A can be obtained.

On the other hand, any vector DNA can be used in the present invention, including plasmid vector DNA and bacteriophage vector D.
NA and the like can be mentioned, and specifically, for example, plasmids pUC118, pUC119, pBLUESCRIPT.
II (STRATAGENE) DNA and the like are preferable. A restriction enzyme such as Bam HI (manufactured by Takara Shuzo Co., Ltd.) is allowed to act on the above vector DNA for digestion to obtain a cut vector DNA.

Then, the DNA containing the growth hormone gene derived from the silver fox and the cleaved vector DNA are mixed, and T4 DNA ligase (manufactured by Boehringer Mannheim) is allowed to act on this to obtain a recombinant DNA. Using this recombinant DNA, for example, Escherichia coli K-12, preferably Escherichia coli JM109 (obtained from Takara Shuzo), XL1-Blue (obtained from Funakoshi) and the like are transformed to obtain the cells of the husband and wife. This transformation can be performed by the method of DM Morrison (Methods in Enzymology, vol.68, 326-331, 1979).

Then, the nucleotide sequence of this growth hormone gene is determined by the method shown in item 6 of the below-mentioned example to determine the nucleotide sequence represented by SEQ ID NO: 1 and translate from the nucleotide sequence. The amino acid sequence represented by SEQ ID NO: 2 was determined.

[0013]

INDUSTRIAL APPLICABILITY The present invention provides a silver fox growth hormone gene and a recombinant DNA containing the same. By culturing, for example, a microorganism containing this recombinant DNA in a medium, silver fox growth hormone can be efficiently produced within an extremely short time, and the present invention is extremely useful industrially. is there.

[0014]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the technical scope of the present invention is not limited by these examples. Example Cloning of Silver Fox Growth Hormone c-DNA (1) Acquisition of Pituitary Tissue Pituitary tissue was immediately removed from 30 freshly slaughtered Silver Fox (obtained from Zao Mink Ranch) to obtain 0.6 g of pituitary tissue. Got This was immediately immersed in liquid nitrogen and frozen and stored in a -80 ° C deep freezer (manufactured by Ebara Co., Ltd.). (2) Acquisition of RNA From 50 mg of pituitary gland obtained in Item 1, Molecular Cloning, 7.19-7.22, Cold Spring Harbor Laboratory
Bor Laboratory) (1989)
μg of total RNA was obtained. (3) Synthesis of c-DNA The synthesis of c-DNA was carried out using a kit manufactured by Boehringer. RNA obtained as described above
Using 60 μg, the Boehringer directed gene (Gen
As a result of treatment according to the method described in e), Vol. 25, p. 263 (1983), 5 μg of double-stranded c-DNA was obtained. (4) Preparation of c-DNA library c-DNA library was prepared by Amersham cDNA
It was performed using a cloning system λgt10, adapter method kit.

1 μ of c-DNA obtained as described above
As a result of carrying out according to the method instructed by Amersham using g, about 2 million cDNA libraries were obtained. (5) Search for Growth Hormone c-DNA Fragments Growth hormone c-DNA was searched according to plaque hybridization selection [Amersham, Hybond blotting membrane protocol]. The details will be described below.

The probe used for plaque hybridization selection is based on mink c-DNA.
DuPont random primer extension labeling system, NEP-103 (RNDOM PRIMER EXTENSION LA
BELING SYSTEM, NEP-103) was used. Using this probe, according to plaque hybridization selection [Hybond blotting membrane protocol manufactured by Amersham, Inc.]
The 10,000 clones of the c-DNA library were searched for silver fox growth hormone c-DNA, and one positive clone was obtained. (6) Analysis of silver fox growth hormone gene The phage DNA of the isolated positive clone was analyzed by the conventional method (M
olecular Cloning, ed. Maniatis et al., p.77-85, Col
d Spring Harbor Laboratory, USA, 1982), cleaved with restriction enzymes such as Bam HI and Eco RI, and examined by the aforementioned electrophoresis. As a result, it was considered that the silver fox growth hormone gene was present on a DNA fragment of about 800 bp which was revealed by cleavage with Bam HI. Therefore, this fragment was subjected to agarose electrophoresis and GENECLEA
Purification by N II (purchased from Funakoshi Corporation), the resulting fragment was inserted into pUC119 cut with Bam HI to obtain recombinant plasmid PfGH300 (see FIG. 1). In addition, this recombinant plasmid was transformed into XL1-Blue according to a conventional method, and E. coli XL1-Blue (pFGH
I got 300). Escherichia coli E1-coli XL1-Blue (pFGH300) has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, as Micro Engineering Research Article No. 4063 (FERM BP-4063). The nucleotide sequence of the obtained plasmid was determined using a Kilo-Sequence deletion kit (purchased from Takara Shuzo) and 370 DNA Sequencing System (purchased from Applied Biosystems). The determined nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide translated from the nucleotide sequence is shown in SEQ ID NO: 2. The silver fox growth hormone gene has a coding region of 648 bases and 216
Coded for one amino acid.

[0017]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 648 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: mRNA to cDNA Origin organism name: Silver fox (Vulpes vulpes) Sequence: ATG GCT GCA AGC CCT CGG AAC TCT GTG CTC CTG GCC TTC GCC TTG 45 CTC TGC CTG CCC TGG CCT CAG GAG GTG GGC GCC TTC CCG GCC ATG 90 CCC TTG TCC AGC CTG TTT GCC AAC GCC GTG CTC CGG GCC CAG CAC 135 CTG CAC CAA CTG GCT GCC GAC ACC TAC AAA GAG TTT GAG CGG GCG 180 TAC ATC CCC GAG GGA CAG AGG TAC TCC ATC CAG AAC GCG CAG GCC 225 GCC TTC TGC TTC TCG GAG ACC ATC CCG GCC CCC ACG GGC AAG GAC 270 GAG GCC CAG CAG CGA TCC GAC GAC GAG CTG CTC CGC TTC TCC CTG 315 CTG CTC ATC CAG TCG TGG CTC GGG CCC GTG CAG TTT CTC AGC AGG 360 GTC TTC ACC AAC AGC CTG GTG TTC GGC ACC TCA GAC CGA GTC TAC 405 GAG AAG CTC AAG GAC CTG GAG GAA GAC CAA GCC CTG ATG CGG 450 GAG CTG GAA GAT GGC AGT CCC CGG GCC GGG CAG ATC CTG AAG CAG 495 ACC TAC GAC AAG TTT GAC ACG AAC CTG CGC AGT GAC GAT GCG CTG 540 CTT AAG AAC TA C GGG CTG CTC TCC TGC TTC AAG AAA GAC CTG CAT 585 AAG GCC GAG ACG TAC CTG CGG GTC ATG AAG TGT CGC CGC TTC GTG 630 GAA AGC AGC TGT GCC TTC SEQ ID NO: 2 Sequence length: 216 Sequence type: Amino acid Topology: Unknown Sequence type: Protein Origin: Organism: Silver Fox Sequence: Met Ala Ala Ser Pro Arg Asn Ser Val Leu Leu Ala Phe Ala Leu 1 5 10 15 Leu Cys Leu Pro Trp Pro Gln Glu Val Gly Ala Phe Pro Ala Met 20 25 30 Pro Leu Ser Ser Leu Phe Ala Asn Ala Val Leu Arg Ala Gln His 35 40 45 Leu His Gln Leu Ala Ala Asp Thr Tyr Lys Glu Phe Glu Arg Ala 50 55 60 Tyr Ile Pro Glu Gly Gln Arg Tyr Ser Ile Gln Asn Ala Gln Ala 65 70 75 Ala Phe Cys Phe Ser Glu Thr Ile Pro Ala Pro Thr Gly Lys Asp 80 85 90 Glu Ala Gln Gln Arg Ser Asp Val Glu Leu Leu Arg Phe Ser Leu 95 100 105 Leu Leu Ile Gln Ser Trp Leu Gly Pro Val Gln Phe Leu Ser Arg 110 115 120 Val Phe Thr Asn Ser Leu Val Phe Gly Thr Ser Asp Arg Val Tyr 125 130 135 Glu Lys Leu Lys Asp Leu Glu Glu Gly Ile Gln Ala Leu Met Arg 140 145 150 Glu Leu Glu Asp Gly Ser Pro Arg Ala Gly Gln Ile Leu Lys Gln 155 160 165 Thr Tyr Asp Lys Phe Asp Thr Asn Leu Arg Ser Asp Asp Ala Leu 170 175 180 Leu Lys Asn Tyr Gly Leu Leu Ser Cys Phe Lys Lys Asp Leu His 185 190 195 Lys Ala Glu Thr Tyr Leu Arg Val Met Lys Cys Arg Arg Phe Val 200 205 210 Glu Ser Ser Cys Ala Phe 215

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme cleavage map of recombinant plasmid pFGH300.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Motoaki Umezu 50-68 Kashima, Matsumori, Izumi-ku, Sendai City, Miyagi Prefecture

Claims (3)

[Claims] 1. A silver fox growth hormone gene encoding the polypeptide represented by SEQ ID NO: 2. 2. A silver fox growth hormone gene represented by SEQ ID NO: 1. 3. A recombinant DNA in which the silver fox growth hormone gene according to claim 1 or 2 is inserted into a vector DNA.