JP2787729B2 - Recombinant mink growth hormone gene, recombinant mink pre growth hormone gene, mink growth hormone or mink pre growth hormone transforming plasmid, and mink growth hormone or mink pre growth hormone transformed Escherichia coli - Google Patents

Description

The present invention relates to mink growth hormone,
In particular, recombinant mink growth hormone, recombinant mink growth hormone structural gene, recombinant mink pre growth hormone, recombinant mink pre growth hormone structural gene, recombinant plasmid, recombinant mink growth hormone and recombinant mink pre growth It relates to a method for producing hormones.

[Background Art] Growth hormone is a peptide hormone produced in pituitary eosinophilic cells.

Mammalian growth hormone is produced in the anterior pituitary gland, but their activity as well as structure is, for example, in humans,
It is already known for cattle, pigs, sheep and the like. There are significant species differences in the biological activity of this hormone, and it has been found that only primates are effective in primates. Also, there are species differences in immunology, and antibodies against human growth hormone do not show cross-reactivity with those of bovine.

As its name implies, the main physiological function of growth hormone is to act on many living tissues and organs of the body to promote growth. It is known that, in addition to promoting growth, it also affects anabolic actions such as protein anabolic action and lipid metabolism.

It has previously been known that administration of growth hormone to animals significantly increases the growth rate, weight gain and meat production of the animals. However, natural growth hormone is contained only in a trace amount in the pituitary gland, and the development of genetic recombination technology has attempted to determine its structure and produce recombinant hormones in microorganisms from humans and cows from an early stage. Was.

[Problems to be Solved by the Invention] However, regarding mink growth hormone, there has been no report that a hormone protein has been isolated, and at present, the structures of the hormone protein and the gene have not been analyzed at all. As mentioned above, there is a remarkable species difference in the biological activity of growth hormone. Therefore, research and development of mink growth hormone have been desired in mink cultivation.

In view of the above problems, the present inventors have performed a structural analysis of mink growth hormone using genetic recombination technology, and have completed the present invention for the purpose of mass supply in the future.

Furthermore, according to the present invention, not only is the cloning of the mink growth hormone structural gene and the nucleotide sequence determined, but also the amino acid sequence of mink growth hormone is elucidated, and the production of mink growth hormone by microorganisms such as Escherichia coli becomes possible. In addition, it is useful for studying more detailed physiological effects of mink growth hormone, which has various physiological effects, and it is expected that its application range will be expanded.

That is, regarding the mink growth hormone gene, the present inventors isolated the mink pre-growth hormone gene, determined the structure thereof, and clarified the structure of the mink growth hormone gene.

[Means for Solving the Problems] The recombinant mink growth hormone structural gene according to the first invention of the present invention encodes the amino acid sequence of the following polypeptide.

The recombinant mink pre-growth hormone structural gene according to the second invention encodes the amino acid sequence of the following polypeptide.

The recombinant plasmid pKS-mGH59 according to the third invention contains a recombinant mink growth hormone structural gene encoding the amino acid sequence of the polypeptide according to the first or second invention.

The E. coli FERM P-11643 transformed with mink growth hormone or mink pre-growth hormone according to the fourth invention is characterized in that
In this invention, the recombinant plasmid pKS-mGH59 described in the above invention was introduced into host E. coli JM109 and transformed. The pituitary tissue extracted from the mink is crushed in a guanidium thiocyanate solution, the RNA fraction is collected by centrifugation, and the obtained RNA fraction is subjected to poly (DNA synthesis) using Oligotex dT30 (manufactured by Nippon Synthetic Rubber Co., Ltd.). A) Poly (A) ⁺ RNA (mRN
A) was concentrated. CDNA was obtained from the mRNA using a cDNA synthesis kit (Pharmacia). Then you got
Plasmid pU with EcoR I end dephosphorylated cDNA
Linked to C19, the mink growth hormone structural gene (cDN
The DNA strand containing A) was completed as a plasmid having a complete circular DNA structure.

The present inventors introduced the obtained plasmid pKS-mGH59 into Escherichia coli JM109 strain, cultured at 37 ° C., and grown pigs on an agar medium containing ampicillin. A plurality of positive colonies were obtained by performing colony hybridization as a probe of a 432 base pair DNA fragment obtained by the Rsa I and Pvu II treatments.

FIG. 1 shows the entire nucleotide sequence of the mink growth hormone structural gene first revealed by the present invention, and the polypeptide consisting of the 190 amino acid sequence shown in claim 2 and claim 3. It contains a polypeptide consisting of the indicated 216 amino acid sequences. From the structure, 26 amino acids, which are considered to be a signal peptide part, were present on the N-terminal side, and the structure of mink growth hormone was elucidated for the first time in the present invention.

Escherichia coli (JM109 strain) into which the present plasmid pKS-mGH59 was introduced as a transformant was deposited under Accession No.
No. 43 has been deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology.

It is further added that the mink growth hormone structural gene and the mink pre-growth hormone structural gene of the present invention can be used in a suitable host (for example, Escherichia coli, Bacillus subtilis, yeast, etc.) to utilize the mink growth hormone or mink growth hormone-like gene. It means that the polypeptide can be produced. That is, a gene sequence capable of regulating the gene obtained in the present invention in an appropriate host (for example,
When a recombinant plasmid containing a promoter called tac, trp, λPL) is introduced and transformed, the production of the polypeptide is under the control of the promoter, and the production can be artificially regulated. That's what it means.

[Action] The growth hormone and derivatives thereof according to the present invention can promote the growth of cultured mink.

EXAMPLES Specific examples of the present invention will be described below.

Example 1. Isolation of total poly (A) + RNA from mink pituitary gland: Isolation of poly (A) + RNA from mink pituitary gland was performed by the guanidium thiocyanate method (Molecular Cloning, 2nd ed). , 1989).

870 mg (approximately 40 individuals) of frozen pituitary gland of 4 min guanidinium thiocyanate (manufactured by Wako Pure Chemical Industries, Ltd. (hereinafter abbreviated as “Wako”)), 0.1 M Tris-HCl (pH 7.5), 1%
It was crushed and solubilized in a 5 ml β-mercaptoethanol solution using a homogenizer. This homogenate was passed through an 18G injection needle several times to separate chromosomal DNA. This solution was diluted with 5.7M cesium chloride (Wako) and 0.01M EDTA (pH
The solution was gently overlaid on the solution of 7.5) so as to be about twice the amount of this solution, and centrifuged at 26,000 rpm for 12 hours with an RPS27-3 rotor manufactured by Hitachi Koki Co., Ltd. to obtain an RNA precipitate. The precipitate is carefully washed with 70% ethanol and 10 mM Tris-
After suspending in HCl (pH 8.0) and 1 mM EDTA solution, ethanol precipitation was performed. The obtained precipitate is suspended in 40 μl of distilled water,
20 μl of the solution was added to 10 mM Tris-HCl, 1 mM EDTA, 0.1% S
After mixing with 100 μl of DS solution, Oligotex dT30
The mixture was mixed with 100 μl of a gel solution (manufactured by Nippon Synthetic Rubber Co.).

After heating this solution at 65 ° C for 5 minutes, it is quenched in ice and 5M N
22 μl of aCl was added. Furthermore, after keeping this solution at 37 ° C. for 10 minutes, the gel to which poly (A) + RNA was adsorbed was precipitated by centrifugation, the supernatant was removed, and then 10 mM Tris-HCl (pH 7.
5) Resuspend in 100 μl of 1 mM EDTA, 0.1% SDS solution,
After heating for 5 minutes, the mRNA was dissociated into the supernatant by centrifugation. After the obtained mRNA was precipitated with ethanol, it was dissolved in 20 μl of distilled water and used for cDNA synthesis.

Example 2. Synthesis of cDNA and integration into vector plasmid: Synthesis of cDNA and integration into a vector were performed using a cDNA synthesis kit manufactured by Pharmacia as follows.

20 μl of the mRNA (2 μg) solution prepared in Example 1 was added to 65
After heating at 10 ° C. for 10 minutes, the mixture was rapidly cooled in ice, added to the first strand reaction mix solution together with 1 μl of the DDT solution, and reacted at 37 ° C. for 1 hour. Add this solution to the second strand reaction mix solution
The reaction was carried out at 12 ° C. for 1 hour and subsequently at 22 ° C. for 1 hour. next,
After adding 1 μl of Klenow fragment and reacting at 37 ° C. for 30 minutes, phenol / chloroform treatment was performed, and the upper layer was purified by a spa column. Obtained cDNA
2 μl of EcoR I / Not I adapter solution and 1 ATP solution
μl and 3 μl of a T4 DNA ligase solution were added and reacted at 12 ° C. overnight, followed by phenol / chloroform treatment and purification with a span column.

The cDNA prepared as described above was ligated to 0.5 μg of a plasmid pUC19 having a dephosphorylated EcoR I end to prepare a plasmid library containing a mink growth hormone gene.

Example 3 Selection of Mink Growth Hormone cDNA Recombinant Plasmid and Nucleotide Sequence of cDNA Portion The recombinant plasmid library obtained as described above was transformed into E. coli JM109 strain by the PEG method (Chung et al., Proceeding of National Co., Ltd.). Transformed according to Academy of Science (Proc. Natl. Acad. Sci.), USA, 86, 2172 (1989), and fixed about 1,200 colonies showing ampicillin resistance to nitrocellulose membrane (Amersham). The porcine growth hormone cDNA was labeled with a DNA fragment consisting of 435 base pairs obtained by treatment with the restriction enzymes Rsa I and Pvu II with horseradish peroxidase (Amersham), and this was used as a probe for colony hybridization. As a result, four positive colonies were obtained.

When a recombinant plasmid was extracted from these colonies and analyzed by restriction enzymes, insertion of a similar DNA fragment was confirmed in all of them. The result is shown in FIG. Then, pKS-mGH59, which was a clone estimated to be almost the full-length cDNA portion, was subjected to the dideoxy method (Sanger et al., Proceeding of National Academy of Science (Proc. Natl. Acad. Sci. ).
USA, 74,5463 (1977)), and the base sequence of mink growth hormone was determined. FIG. 1 shows the nucleotide sequence and amino acid sequence of the cDNA portion.

[Effects of the Invention] As described above, the present invention is based on the amino acid sequence of mink growth hormone firstly elucidated by the present invention, and includes various amino acid sequences including a deoxyribonucleotide chain encoding all or a part of the amino acid sequence. Expression vectors are constructed, and each is introduced into Escherichia coli, Bacillus subtilis, photosynthetic cells, yeast or animal cells, etc. as appropriate transformants, and then cultured to facilitate the production of recombinant mink growth hormone from various transformants. In addition, there is an effect that a large amount can be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing the entire nucleotide sequence of the complementary strand DNA portion containing mink growth hormone and the amino acid sequence revealed from the sequence, and FIG. 2 is a restriction enzyme cleavage map of the portion containing mink growth hormone complementary chain DNA.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:19) (C12P 21/02 C12R 1:19) (56) References JP-A-59-144743 (JP, A) JP-A-57-171999 (JP, A) JP-A-60-11557 (JP, A) JP-A-1-173873 (JP, A) Biochemica et Biophysica Acta, 1048 [2-3] (1990-4-6) P.). 290-293 (58) Fields investigated (Int. Cl. ⁶ , DB name) WPI (DIALOG) BIOSIS (DIALOG) CA (STN) REGISTRY (STN) GenBank / EMBL / DDBJ (GENETYX) (54) Name: Recombinant mink growth hormone gene, recombinant mink pre-growth hormone gene, mink growth hormone or mink pre-growth hormone transforming plasmid, and mink growth hormone or mink pre-growth hormone transformed E. coli

Claims (4)

(57) [Claims] 1. A recombinant mink growth hormone structural gene encoding the amino acid sequence of the following polypeptide. 2. A recombinant mink pre-growth hormone structural gene encoding the amino acid sequence of the following polypeptide. 3. A recombinant plasmid pKS-mGH5 containing a recombinant mink growth hormone structural gene encoding the amino acid sequence of the polypeptide according to claim 1 or 2.
9. 4. The recombinant plasmid pKS-m according to claim 3.
Mink growth hormone or mink pre-growth hormone-transformed Escherichia coli FERM P-11643 transformed by introducing GH59 into host Escherichia coli JM109.