JPH07155183A - Recombinant plasmid vector, bacterium of genus bacillus and production of porcine growth hormone - Google Patents

Abstract

PURPOSE:To efficiently obtain a large amount of a natural type porcine growth hormone at a low cost in a simple step. CONSTITUTION:This recombinant plasmid vector contains a hybrid porcine growth hormonal gene composed of a genetic region coding for as signal sequence of a protein secreted by a bacterium of the genus Bacillus such as Bacillus subtilis alpha-amylase, a structural genetic region of the porcine growth hormone located on the down stream side thereof and a promoter capable of functioning with a bacterium of the genus Bacillus such as the Bacillus subtilis on the upstream side of the hybrid porcine growth hormonal gene. A bacterium of the genus Bacillus such as a low-protease Bacillus subtilis is transformed by this vector and this method for producing a large amount of porcine growth hormone is to culture the resultant transformant.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant plasmid vector containing a gene encoding porcine growth hormone, a bacterium belonging to the genus Bacillus transformed with this vector, and a method for producing porcine growth hormone by the bacterium belonging to the genus Bacillus.

[0002]

BACKGROUND OF THE INVENTION Porcine growth hormone is secreted from porcine pituitary eosinophilic cells and has a molecular weight of about 2200 consisting of 191 amino acid residues.
0 protein. Porcine growth hormone has physiological activities such as a growth promoting action and a milk amount increasing action,
It can be used as a growth promoter for pigs.

On the other hand, as a method of producing pig growth hormone, a method of extracting from pig pituitary is known. However, this method has low productivity.

A method for producing porcine growth hormone by Escherichia coli using a gene recombination technique is also known.
However, this method has the following problems. That is, since the translation initiation codon ATG is required in the 5'region of the gene, an extra methionine residue is bound to the N-terminus of the produced hormone protein, and a methionyl-
Pig growth hormone is produced. In Escherichia coli, since intracellular production is carried out, 2
No cysteine-cysteine bond is formed. Therefore, the product cannot be used as it is, and a reaction step for removing a methionine residue and forming a cysteine-cysteine bond is required. Furthermore, in the method using Escherichia coli as a host, a large amount of pyrogen is mixed with porcine growth hormone. Therefore, a large amount of purification equipment is required.

Therefore, an object of the present invention is to provide a recombinant plasmid vector which can directly and efficiently produce a safe natural type porcine growth hormone which can be directly applied to pigs.

Another object of the present invention is to provide a bacterium belonging to the genus Bacillus which is transformed with the above vector and secretes and produces a large amount of natural porcine growth hormone.

Still another object of the present invention is to provide a method for producing porcine growth hormone, which can efficiently obtain natural porcine growth hormone in a simple process, in large quantities and at low cost.

[0008]

The present inventors have conducted extensive studies focusing on Bacillus bacteria which have excellent safety and the ability to secrete and produce large amounts of proteins outside the cells. As a result, the inventors secreted a protein secreted by Bacillus bacteria. When a recombinant vector having a gene encoding the signal sequence of Escherichia coli (signal peptide) and a gene encoding the mature protein of porcine growth hormone was constructed and the Bacillus bacterium transformed with this vector was cultured, an extra N-terminal was produced. The present invention has been completed by finding that natural porcine growth hormone in which a cysteine-cysteine bond is formed in the molecule is efficiently secreted and produced in large amounts without any methionine residue.

That is, the present invention provides (1) a hybrid pig growth hormone gene comprising a gene region encoding a signal sequence of a Bacillus bacterial secretory protein and a pig growth hormone structural gene region located downstream thereof; A recombinant plasmid vector containing a promoter capable of functioning in Bacillus bacteria upstream of the porcine growth hormone gene,
(2) A bacterium belonging to the genus Bacillus transformed with the recombinant plasmid vector, and (3) a method for producing pig growth hormone, which comprises culturing the bacterium belonging to the genus Bacillus and recovering the produced pig growth hormone.

The signal sequence of the Bacillus bacterial secretory protein may be a signal sequence of a protein secreted by Bacillus bacteria such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and the like. Examples thereof include signal sequences of α-amylase, alkaline protease, neutral protease, levansucrase and the like. Among the above signal sequences, the signal sequence of Bacillus subtilis α-amylase is preferably used.

The gene encoding the signal sequence is
It functions efficiently in Bacillus bacteria. These genes may be prepared from the gene encoding the protein, or may be chemically synthesized by determining the base sequence from the amino acid sequence of the signal sequence portion of the protein.

The gene encoding the signal sequence is
It has a translation initiation codon ATG at the 5'end. The signal sequence is cleaved and removed when porcine growth hormone is secreted and produced by Bacillus bacterium. Therefore, when a Bacillus bacterium transformed with the recombinant plasmid vector of the present invention is cultured, a natural porcine growth hormone in which an extra methionine residue is not bound to the N-terminus is produced.

The porcine growth hormone structural gene may be any gene that encodes porcine growth hormone, and is either the porcine growth hormone gene obtained from pigs or the gene chemically synthesized based on the amino acid sequence of porcine growth hormone. May be. Examples of the porcine growth hormone structural gene include genes having the following base sequences.

[0014] 1 TTTCCTGCCA TGCCGCTGTC ATCCCTTTTC AAAGGACGGT ACGGCGACAG TAGGGAAAAG 31 GCTAACGCAG TTTTACGTGC TCAACATTTA CGATTGCGTC AAAATGCACG AGTTGTAAAT 61 CACCAGCTTG CAGCGGATAC ATATAAAGAA GTGGTCGAAC GTCGCCTATG TATATTTCTT 91 TTTGAAAGAG CTTACATTCC AGAAGGCCAA AAACTTTCTC GAATGTAAGG TCTTCCGGTT 121 CGCTATTCTA TCCAGAACGC ACAAGCGGCT GCGATAAGAT AGGTCTTGCG TGTTCGCCGA 151 TT CTGCTTTA GCGAGACGAT TCCTGCACCG AAGACGAAAT CGCTCTGCTA AGGACGTGGC 181 ACAGGCAAAG ATGAAGCTCA GCAACGTTCT TGTCCGTTTC TACTTCGAGT CGTTGCAAGA 211 GACGTTGAAC TGCTTAGATT CTCTTTACTG CTGCAACTTG ACGAATCTAA GAGAAATGAC 241 CTTATCCAAA GCTGGCTGGG TCCAGTACAG GAATAGGTTT CGACCGACCC AGGTCATGTC 271 TTCTTATCTC GAGTATTTAC TAATAGCTTG AAGAATAGAG CTCATAAATG ATTATCGAAC 301 GTGTTTGG A CCAGTGACCG TGTCTACGAG CACAAACCAT GGTCACTGGC ACAGATGCTC 331 AAATTAAAGG ATCTGGAAGA AGGCATTCAA TTTAATTTCC TAGACCTTCT TCCGTAAGTT 361 GCATTAATGC GTGAGTTAGA AGATGGAAGC CGTAATTACG CACTCAATCT TCTACCTTCG 391 CCTCGTGCAG GCCAAATCTT GAAACAGACT GGAGCACGTC CGGTTTAGAA CTTTGTCTGA 421 TACGACAAAT TCGACACGAA CCTTCGCTCT ATGCTGTTTA AGCTGTGCTT GGAAGCGAGA 451 GATGATGCGC TGTTG AAGAA CTATGGATTA CTACTACGCG ACAACTTCTT GATACCTAAT 481 CTCTCCTGTT TTAAAAAGGA TCTTCATAAA GAGAGGACAA AATTTTTCCT AGAAGTATTT 511 GCCGAAACAT ATTTGCGTGT AATGAAATGT CGGCTTTGTA TAAACGCACA TTACTTTACA 541 CGTCGCTTCG TTGAGAGCTC CTGCGCTTTT GCAGCGAAGC AACTCTCGAG GACGCGAAAA in such base sequence is high genetic codons are used with many expression efficiency is frequently used in B. subtilis Because
The production amount of porcine growth hormone can be significantly improved.

The hybrid porcine growth hormone gene is not particularly limited as long as the porcine growth hormone structural gene is located at the downstream side of the gene encoding the signal sequence.

The promoter capable of functioning in the Bacillus bacterium includes a gene encoding a protein secreted by the Bacillus bacterium such as Bacillus subtilis, for example, α-amylase, alkaline protease, neutral protease, levansucrase and the like. A promoter is mentioned. These promoters can function efficiently in Bacillus bacteria. As the promoter, particularly α of Bacillus subtilis
-A promoter for the amylase gene is preferably used.

The promoter can be obtained by cloning it by a known method or by preparing it from a plasmid, but it can also be obtained by chemical synthesis based on the amino acid sequence. The promoter is, for example, the plasmid pUC11.
8 inserted between EcoRI and XbaI, and can be used by cutting and recovering as a BamHI-XbaI fragment from the above plasmid at the time of ligation to the expression vector of porcine growth hormone gene.

By ligating the hybrid porcine growth hormone gene downstream of the promoter, it becomes possible to produce porcine growth hormone in Bacillus bacteria.

The recombinant plasmid vector of the present invention comprises:
It is preferable that SD (Shine-Dalgarno) sequences are linked. The SD sequence is usually linked between the hybrid porcine growth hormone gene and the promoter. The SD sequence is not particularly limited, and for example, 16S-rRN of Bacillus subtilis
An SD sequence synthesized corresponding to the base sequence of A can be used. The SD sequence functions as a ribosome binding site.

In the recombinant plasmid vector of the present invention, downstream of the hybrid pig growth hormone gene,
A terminator may be connected. The terminator is not particularly limited, and may be, for example, a terminator of an alkaline protease gene of Bacillus subtilis, or a terminator chemically synthesized corresponding thereto. By connecting a terminator, expression of a structural gene can be increased.

The vector used for constructing the recombinant plasmid vector of the present invention may be any vector so long as it can be stably replicated in Bacillus bacterium such as Bacillus subtilis. For example, plasmid pUB110, pC
194, pE194, pTHT15, pBD16 and derivatives thereof and the like.

The construction of the recombinant plasmid vector of the present invention is carried out by "Molecular Cloning"("MolecularClon").
ing ”),“ A Laboratory Manual ”(“ A Labo
ratory Mannual ”, Cold Spring Harbor Laboratory, 1
It can be carried out by a known method described in 989). For example, after ligating the promoter, SD sequence, signal sequence gene, porcine growth hormone structural gene and terminator on an Escherichia coli vector, a cleavage fragment containing all the gene fragments can be stably replicated in Bacillus bacteria. The recombinant plasmid vector of the present invention can be constructed by introducing it into the inside.

More specifically, the recombinant plasmid vector can be constructed by the following method, for example.

(1) Porcine growth hormone structural gene (EcoRI having a connecting site with the signal sequence gene on the upstream side)
-BamHI fragment) is prepared and ligated between EcoRI-BamHI of a suitable E. coli plasmid. An appropriate host is transformed with this plasmid, and a porcine growth hormone structural gene-introduced plasmid is prepared from the obtained transformant.

At this time, the porcine growth hormone structural gene may be synthesized as a plurality of fragments, and each fragment may be introduced into a plurality of plasmids.

For example, the porcine growth hormone structural gene
It is synthesized as 6 DNA fragments (1st to 26th fragments), and a ligation site with the signal sequence gene is provided on the upstream side of the most upstream fragments (25th and 26th fragments). After treatment such as quenching and annealing by a conventional method, the downstream first to twelfth fragment portions (BamHI
-KpnI fragment), for example E. coli plasmid pUC
Between BamHI-KpnI of 19 upstream thirteenth to second
The 6 fragment portion (KpnI-EcoRI fragment) is ligated between KpnI-EcoRI of the plasmid pUC19, and E. coli JM109 strain is transformed, for example. From the ampicillin-resistant transformant, a plasmid having the 1st to 12th fragments and a plasmid having the 13th to 26th fragments introduced therein can be prepared.

(2) On the other hand, the promoter, SD sequence, signal sequence gene and terminator are prepared by the method described above.

(3) A plasmid capable of stable replication in Bacillus bacteria, for example, S of Bacillus subtilis plasmid pUB110.
The phI-EcoRI fragment was added to the Escherichia coli vector, eg, the polylinker site SphI-of plasmid pUC19.
Introduce between EcoRI. Bam of the obtained plasmid
The promoter, SD sequence, signal sequence gene, porcine growth hormone structural gene excised from the plasmid obtained in (1) above and terminator are ligated to the HI cleavage site sequentially or simultaneously, and, for example, Escherichia coli is transformed. From the obtained transformant, an Escherichia coli plasmid containing all of the above gene fragments is obtained.

(4) SphI of the Escherichia coli plasmid
A plasmid capable of stably replicating the AvaI fragment in a Bacillus bacterium, for example the Bacillus subtilis plasmid pUB1 described above.
The recombinant plasmid vector of the present invention can be obtained by ligating to the cleavage sites of 10 with SphI and AvaI. Said SphI-AvaI fragment may be a plurality of fragments, for example SphI-KpnI fragment and KpnI-.
You may collect and use it as an AvaI fragment.

By transforming a bacterium belonging to the genus Bacillus with the recombinant plasmid vector obtained as described above, a microorganism producing a large amount of porcine growth hormone can be obtained. Transformation of a Bacillus bacterium with the above vector is carried out by a conventional method, for example, a protoplast transformation method with PEG6000 [Chang, S., et al., Mol. Ge.
n. Genet., 168, 111-115 (1979)] and the like.

The above-mentioned host microorganism is Bacillus
The strain is a microorganism of the genus, and its strain is not particularly limited as long as the gene product is stable. Unlike Escherichia coli and the like, Bacillus bacteria are extremely safe microorganisms because they do not produce endotoxin and do not parasitize the human body. In addition, it has the ability to secrete and produce proteins outside the cells, and it has been used for a long time in Japan for the production of natto, and also as an industrial production bacterium of enzymes, amino acids, antibiotics, etc. It is a very easy-to-use microorganism.

Among the Bacillus bacteria, a preferred host is
It is a highly safe Bacillus subtilis ( Bacillus subtilis ) that secretes a large amount of protein outside the cells. Further, among Bacillus subtilis, strains having reduced protease activity outside the cells by a method such as mutation are preferable. When such a strain is transformed, decomposition by a protease derived from a host can be suppressed, which is particularly advantageous for the production of porcine growth hormone.

As Bacillus subtilis having a low protease activity,
For example, Bacillus subtilis 104HL strain deficient in both the neutral protease and the alkaline protease, which are the main extracellular proteases of Bacillus subtilis [Biochem. Biophys. Res. Commun. ), 128;
601-606, 1985]; Bacillus subtilis DY-1 in which pap gene was introduced into Bacillus subtilis 104HL strain
6 strains (Microtech Lab. No. 9488); Bacillus subtilis lacking the ability to produce alkaline protease and neutral protease and having protease activity of 3% or less of the wild strain, spo OA △ 67
7 Examples include strains into which a mutant gene has been introduced. Particularly preferred strains include, for example, Bacillus subtilis 104.
Bacillus that has introduced the spo OA Δ677 mutant gene into the HL strain
Subtilis SPO11 strain
No.), Spo OA △ 67 to Bacillus subtilis DY-16 strain
7 Mutant gene-introduced Bacillus subtilis SPL1
4 strains (Microtechnology Research Institute, No. 10988) and the like are included (see JP-A-3-143387).

Particularly, the Bacillus subtilis SPL14 strain has a protease activity of 1 / 5,000 or less as compared with the wild strain, has extremely good stability of the product, has two kinds of amino acid requirements, and does not form spores. Therefore, since it is considered that it cannot grow in the natural world, it is an extremely useful bacterium as a host for industrial production of porcine growth hormone.

Pig growth hormone can be obtained by culturing the transformed Bacillus bacterium. Cultivation of the transformed Bacillus bacterium can be carried out according to conventional liquid culture. That is, the culture of the transformant strain can be carried out by a shaking culture or an aeration and agitation culture method in a liquid medium containing a conventional component such as an inorganic salt, a carbon source, a nitrogen source and a growth factor component. The pH of the medium is, for example, about 7-8. Cultivation can be performed under conditions usually employed for culturing microorganisms, for example, a temperature of 15 to 45 ° C, preferably 25 to 40 ° C, and a culturing time of about 6 to 60 hours.

Thus, a natural porcine growth hormone having no methionine residue bonded to the N-terminal and having a cysteine-cysteine bond formed in the molecule is produced.

The porcine growth hormone produced by the host microorganism can be separated and purified by conventional methods such as centrifugation, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration and S.
DS-agarose gel electrophoresis, SDS-polyacrylamide gel electrophoresis, electroelution, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, or a combination of these methods it can. When porcine growth hormone is accumulated in the cells or periplasm, the porcine growth hormone can be obtained by a conventional method, for example, by crushing the cells and subjecting to separation and purification.

[0038]

INDUSTRIAL APPLICABILITY Since the porcine growth hormone expression vector of the present invention contains a porcine growth hormone structural gene and a signal sequence and a promoter capable of efficiently functioning in Bacillus bacteria, it is effective and safe for pigs as it is. It is possible to directly and efficiently produce natural pig growth hormone in a large amount.

Since the Bacillus bacterium of the present invention is transformed with the excellent vector as described above, a large amount of natural porcine growth hormone is secreted and produced.

The production method of the present invention comprises the step of culturing the Bacillus bacterium as described above, in order to cultivate the natural pig growth hormone,
It can be obtained efficiently, in large quantities, and at low cost by a simple process.

[0041]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited thereto.

Example (1) Synthesis of promoter of α-amylase gene The complementary strands of the promoter of Bacillus subtilis α-amylase gene having the nucleotide sequences shown below were chemically synthesized using a DNA synthesizer (manufactured by ABI). 5'of both genes synthesized
The region was phosphorylated and then annealed to obtain Ec of pUC118.
inserted between oRI-XbaI and plasmid pMD30
I got 03. A construction diagram of pMD3003 is shown in FIG.

[0043] For reference the sequence of the 5'-AATTCAGGATCCTTGATTTGTATTC ACTCTGCCAAGTTGTTTTGATAGAGTGA TTGTGATAATTTAAAATGTAAGCGTGAA CAAAATTCTCCAGT-3 '5'-ACTGGAGAATTTTGTTCACGCTTAC ATTTTAAATTATCACAATCACTCTATCA AAACAACTTGGCAGAGTGAATACAAATC AAGGATCCTG-3' (2) 16S ribosomal RNA synthesis Bacillus subtilis SD sequence (rRNA), with the following nucleotide sequence The represented SD sequences were synthesized. In addition, an XbaI site, which is a connecting site with a promoter, was introduced into the 5 ′ side of the gene, and an NruI site, which is a connecting site with the signal sequence of the α-amylase gene, was introduced into the 3 ′ region. The underlined portion of the following sequence shows the SD sequence.

After purification by a conventional method, the 5'region was phosphorylated with T4 polynucleotide kinase and annealed. Then, subject to polyacrylamide electrophoresis, cut out the desired band, and perform D
The NA fragment was recovered and used as an SD sequence fragment.

CT AGAAAGGAGGGTGATC TAGAATTCATGTTTCG TTTCCTCCACTACATCTTAAGTACAAGGC (3) Synthesis of α-amylase signal sequence gene Amino acid sequence of signal sequence part of Bacillus subtilis α-amylase [Yamazaki, H., et al., J. Bacteriol., 156 , 156 , 156 , 156 , 156 , 156 ,
(1984)], complementary strands of the α-amylase signal sequence gene having the following nucleotide sequences were respectively synthesized.
In addition, an NruI site, which is a connecting site with the SD sequence, was introduced at the 5'end of the gene, and an HpaII site, which is a connecting site with the porcine growth hormone structural gene, was introduced at the 3'side.

After purification by a conventional method, it was phosphorylated and annealed. Then, after being subjected to electrophoresis, it was recovered and used as a synthetic DNA linker.

[0047] As 5'-CGAAACGATTCAAAACCTCTTTACT GCCGTTATTTGCTGGATTTTTATTGCTG TTTTATTTGGTTCTGGCAGGAC-3 '5'-CGGTCCTGCCAGAACCAAATAAAAC AGCAATAAAAATCCAGCAAATAACGGCA GTAAAGAGGTTTTGAATCGTTTCG-3' (4) referring to Taminata alkaline protease gene produced Bacillus subtilis terminator, the complementary strands of the terminator having the nucleotide sequence of the following Each was synthesized. On the 5'end side, B
A glII site was provided and a BamHI site was provided on the 3'end side. After synthesis, the 5'regions of both synthetic genes were phosphorylated and annealed. Then, after being subjected to electrophoresis, it was recovered and used as a terminator fragment.

[0048] 5'-GATCTATAAAAAGAAGCAGGTTCCT CCATACCTGCTTCTTTTTATTTGTCAGC ATCCTGATGTTGGATCCGCATG-3 '5'-CGGATCCAACATCAGGATGCTGACA AATAAAAAGAAGCAGGTATGGAGGAACC TGCTTCTTTTTATA-3' (5) cut out small pieces of about 1 kbp to EcoRI from SphI to construct a plasmid pUB110 of PUC19-110B, plasmid pUC19 Of the polylinker site of SphI-EcoR
Introduced between I to construct pUC19-110B. p
A construction diagram of UC19-110B is shown in FIG.

(6) Synthesis of Porcine Growth Hormone Gene A gene to be synthesized was designed in consideration of the gene codon usage frequency of Bacillus subtilis and the codon usage frequency of Escherichia coli. That is, using gene codons that are frequently used in Bacillus subtilis, both strands of the gene were synthesized as 26 DNA fragments each capable of forming about 50 base pairs. FIG. 3 shows the synthesized DNA fragment of the porcine growth hormone structural gene.

In order to ligate the 5'end of the porcine growth hormone gene with the signal sequence gene of the Bacillus subtilis α-amylase gene, fragments 25 and 26 of the above DNA fragments have the following sequences added upstream. Was synthesized as.

AATTCCGGGCGGCTGCGAGTGCCAGCCGCCGACGCTCACGT In addition, in order to ligate the 3 ′ end of the porcine growth fulmon gene to the BamHI site of the vector, a sequence containing a BamHI recognition site downstream of fragments 1 and 2 of the above DNA fragments was added and synthesized. .

The synthesized DNA fragment was deprotected with ammonia and then purified using an OPC cartridge (manufactured by Applied Biosystems). The purified DNA fragments were each phosphorylated at the 5'site using T4-DNA Kinase.

(7) Construction of Porcine Growth Hormone Gene After annealing the phosphorylated DNA fragment with its complementary strand, 15% polyacrylamide gel electrophoresis (PAGE) was performed to cut out a band of a desired size, It was recovered by the electrophoretic elution method. Recovered double-stranded DN
Mix two or three A's and mix with T4-DNA Liga
It was ligated using se. Then, 12% polyacrylamide gel electrophoresis was performed, a band of a desired size was cut out, and the band was similarly recovered by the electrophoretic elution method.

Among the recovered double-stranded DNAs, the above (6)
D containing fragments 1 to 12 of the DNA fragment synthesized in
NA was added to KpnI-BamHI of plasmid pUC19.
In between, the DNA containing fragments 13-26 was added to plasmid pU
Escherichia coli JM109 strain was transformed by ligating each of C19 between EcoRI and KpnI. A plasmid DNA was prepared from the obtained ampicillin-resistant transformant and subjected to electrophoresis. As a result, clones considered to be the target plasmid were obtained. Plasmid DNA was prepared from the obtained transformant, cleaved with various restriction enzymes, and then subjected to agarose gel electrophoresis to examine whether the desired plasmid was formed. In addition, for the plasmid from which a fragment of the desired size was obtained, insert the DN
The nucleotide sequence was confirmed using A Sequencer 373A (manufactured by Applied Biosystems). As a result, the target plasmids pDPGH11 and pDPGH
I got 12. FIG. 4 shows a construction diagram of plasmids pDPGH11 and pDPGH12.

(8) Ligation of Porcine Growth Hormone Gene to Expression Vector Ligation of the porcine growth hormone (PGH) gene to an expression vector was first carried out in an E. coli system.

The porcine growth hormone gene was transformed into the pDPG
From H12 and pDPGH11, respectively, HpaII
It was digested and recovered as a -KpnI fragment and a KpnI-BamHI fragment. At this time, BamH from pDPGH11
The I breakpoint was treated with alkaline phosphatase and dephosphorylated.

On the other hand, pMD3003 constructed in (1) above
The promoter portion was cleaved and recovered as a BamHI-XbaI fragment. In addition, pUC19 constructed in (5) above
Since -110B was used as a vector, it was cleaved with BamHI, treated with alkaline phosphatase and dephosphorylated at the cleavage point.

PUC19-1 cleaved with BamHI
10B, the above promoter BamHI-XbaI fragment,
SD sequence fragment prepared in (2) above, α-amylase signal sequence gene fragment prepared in (3) above, pDPG
HpaII-KpnI fragment from H12, pDP above
The KpnI-BamHI fragment obtained from GH11 and the terminator fragment prepared in (4) above were used as T fragments.
Ligation was performed using 4 ligase, and Escherichia coli JM109 strain was transformed. When a plasmid DNA was prepared from the obtained transformant resistant to ampicillin and examined, one strain of the target plasmid pDPGH13 was obtained. The nucleotide sequence of the synthetic DNA portion of the obtained plasmid DNA was confirmed. FIG. 5 shows a construction diagram of the plasmid pDPGH13.

(9) PGH to Bacillus subtilis plasmid vector
Ligation of Gene Next, the porcine growth hormone gene was ligated into the Bacillus subtilis plasmid vector pUB110.

First, pUB110 is treated with SphI and AvaI.
It was cut with and the vector portion was recovered. On the other hand, the porcine growth hormone gene was recovered as two fragments of AvaI-KpnI and SphI-KpnI from pDPGH13 obtained in (8) above. These three fragments were ligated and transformed into the low protease Bacillus subtilis host strain SPL14. When a plasmid was recovered from the obtained kanamycin-resistant transformant and examined, the desired plasmid pDPGH3 was obtained.
0 was obtained. FIG. 6 shows a construction diagram of the plasmid pDPGH30.

(10) Bacillus subtilis SPL14 (pDPGH30)
Secretory production of porcine growth hormone by Bacillus subtilis SPL14 (pDPGH30) was cultured as follows, and porcine growth hormone was secreted and produced in the culture supernatant.

First, set SPL14 (pDPGH30) to 1
0 mL of L medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl pH 7.2 15 μg / mL kanamycin) was inoculated and cultured at 37 ° C. overnight. Collect 1 mL of culture broth, collect cells by centrifugation, and collect 0.85% N
Suspension in an aCl aqueous solution was repeated (twice) to wash the bacteria. Next, the bacteria suspended in 0.85% NaCl aqueous solution were mixed with 100
mL LS medium (2% tryptone, 1.0% yeast extract, 0.5% NaCl 1% soluble starch 0.5M
The cells were inoculated into a 500 ml-volume conical Erlenmeyer flask containing sodium succinate (pH 7.3, 150 μg / mL kanamycin), and cultured with shaking using a rotary shaker (125 rpm, 37 ° C.) for 15 hours. The culture solution was centrifuged to obtain a culture supernatant containing porcine growth hormone.

(11) Bacillus subtilis SPL14 (pDPGH30)
Porcine growth hormone secretory productivity of SPL14 (pDPGH30) The amount of porcine growth hormone secreted in the culture supernatant of SPL14 (pDPGH30) was measured by the slot blot method using an anti-porcine growth hormone antibody. For slot blotting, Biodot SF device (manufactured by Japan Biorat Co., Ltd.) was used, and according to the manual attached to the device,
Nitrocellulose membrane (hereinafter abbreviated as membrane)
The sample was blotted on. Sample is SPL
14 (pDPGH30) culture supernatant was added to TBS buffer (20 mM Tris 500 mM NaCl pH 7.
A solution diluted 1 / 20-1 / 160 in 5) and a solution of porcine growth hormone extracted from porcine pituitary gland in TBS buffer were used as standards.

Next, porcine growth hormone on the membrane was detected. First, the membrane on which the sample was blotted was blotting solution (3% gelatin / TBS
Buffer) and gently shaken for 1 hour at room temperature. Membrane is TTBS buffer (0.05% Tw
een20 / TBS) and washed twice (5 minutes x 2)
Then, the primary antibody solution (anti-porcine growth hormone antibody (UCB
1/500 volume / 1% gelatin / TTB
S buffer). After shaking at room temperature for 1 hour,
Transfer to TTBS buffer and wash twice (5 min x 2),
Secondary membrane solution (peroxidase (HRP))
Labeled anti-rabbit IgG antibody (Bio-Rad) 1/30
00 volume / 1% gelatin / TTBS buffer). Membrane shaken for 1 hour with TTBS
After washing twice with the buffer (5 minutes × 2) and 5 minutes in the TBS buffer, HRP was developed and the porcine growth hormone immobilized on the membrane was detected. Color development of HRP was performed using Immunostain HRP kit IS-50B Konica (manufactured by Konica Corp.).

The HRP color intensity of each spot on the membrane was measured with an image analyzer (Model AE-6, manufactured by Atto Co., Ltd.).
900). A calibration curve was prepared from the color development intensity of porcine growth hormone extracted from porcine pituitary gland, and the porcine growth hormone secreted amount of Bacillus subtilis SPL14 (pDPGH30) was measured. As a result, SPL14 (pDPGH3
The strain 0) secreted and produced about 10 mg / L of porcine growth hormone in the culture supernatant.

10 μL of the culture supernatant of the SPL14 (pDPGH30) strain was separated by SDS-polyacrylamide gel electrophoresis according to a conventional method, and then transferred to a nitrocellulose membrane using a transfer device. As a result of detecting porcine growth hormone transferred on the membrane according to the method of (11) above, a band was detected at the same position as that of porcine growth hormone extracted from porcine pituitary gland.

[0067]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 570 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: CDS Location: 1. ． 570 methods to determine the characteristics: S sequence TTTCCTGCCA TGCCGCTGTC ATCCCTTTTC GCTAACGCAG TTTTACGTGC TCAACATTTA 60 CACCAGCTTG CAGCGGATAC ATATAAAGAA TTTGAAAGAG CTTACATTCC AGAAGGCCAA 120 CGCTATTCTA TCCAGAACGC ACAAGCGGCT TTCTGCTTTA GCGAGACGAT TCCTGCACCG 180 ACAGGCAAAG ATGAAGCTCA GCAACGTTCT GACGTTGAAC TGCTTAGATT CTCTTTACTG 240 CTTATCCAAA GCTGGCTGGG TCCAGTACAG TTCTTATCTC GAGTATTTAC TAATAGCTTG 300 GTGTTTGGTA CCAGTGACCG TGTCTACGAG AAATTAAAGG ATCTGGAAGA AGGCATTCAA 360 GCATTAATGC GTGAGTTAGA AGATGGAAGC CCTCGTGCAG GCCAAATCTT GAAACAGACT 420 TACGACAAAT TCGACACGAA CCTTCGCTCT GATGATGCGC TGTTGAAGAA CTATGGATTA 480 CTCTCCTGTT TTAAAAAGGA TCTTCATAAA GCCGAAACAT ATTTGCGTTC540TCTTGAACTGA

[0068]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 95 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: promoter Location: 1. ． 95 Characterization Method: S Sequence AATTCAGGAT CCTTGATTTG TATTCACTCT GCCAAGTTGT TTTGATAGAG TGATTGTGAT 60 AATTTAAAAT GTAAGCGTGA ACAAAATTCT CCAGT 95

[0069]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: binding-site Location: 3. ． 17 Method of characterizing: S sequence CTAGAAAGGA GGTGATCTAG AATTCATGTT CG 32

[0070]

[Sequence list]

 SEQ ID NO: 4 Sequence length: 75 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: sig peptide Location: 1 ． ． 75 Characterized Method: S Sequence CGAAACGATT CAAAACCTCT TTACTGCCGT TATTTGCTGG ATTTTTATTG CTGTTTTATT 60 TGGTTCTGGC AGGAC 75

[0071]

[Sequence list]

 SEQ ID NO: 5 Sequence length: 75 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: terminator Location: 1. ． 75 Characterization Method: S Sequence GATCTATAAA AAGAAGCAGG TTCCTCCATA CCTGCTTCTT TTTATTTGTC AGCATCCTGA 60 TGTTGGATCC GCATG 75

[Brief description of drawings]

FIG. 1 is a construction diagram of plasmid pMD3003.

FIG. 2 is a construction diagram of plasmid pUC19-110B.

FIG. 3 shows a DNA fragment of the porcine growth hormone structural gene.

FIG. 4: Plasmid pDPGH11 and plasmid p
It is a construction drawing of DPGH12.

FIG. 5 is a construction diagram of plasmid pDPGH13.

FIG. 6 is a construction diagram of plasmid pDPGH30.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 125) (C12P 21/02 C12R 1: 125)

Claims (5)

[Claims] 1. A hybrid porcine growth hormone gene comprising a gene region encoding a signal sequence of a Bacillus bacterial secretory protein and a porcine growth hormone structural gene region located downstream thereof, and upstream of the hybrid porcine growth hormone gene. A recombinant plasmid vector containing a promoter capable of functioning in Bacillus bacteria. 2. The Bacillus bacterial secretory protein is Bacillus subtilis α-
The recombinant plasmid vector according to claim 1, which is amylase. 3. The recombinant plasmid vector according to claim 1, wherein the porcine growth hormone structural gene has the following base sequence. 1 TTTCCTGCCA TGCCGCTGTC ATCCCTTTTC AAAGGACGGT ACGGCGACAG TAGGGAAAAG 31 GCTAACGCAG TTTTACGTGC TCAACATTTA CGATTGCGTC AAAATGCACG AGTTGTAAAT 61 CACCAGCTTG CAGCGGATAC ATATAAAGAA GTGGTCGAAC GTCGCCTATG TATATTTCTT 91 TTTGAAAGAG CTTACATTCC AGAAGGCCAA AAACTTTCTC GAATGTAAGG TCTTCCGGTT 121 CGCTATTCTA TCCAGAACGC ACAAGCGGCT GCGATAAGAT AGGTCTTGCG TGTTCGCCGA 151 TTCTGCTT A GCGAGACGAT TCCTGCACCG AAGACGAAAT CGCTCTGCTA AGGACGTGGC 181 ACAGGCAAAG ATGAAGCTCA GCAACGTTCT TGTCCGTTTC TACTTCGAGT CGTTGCAAGA 211 GACGTTGAAC TGCTTAGATT CTCTTTACTG CTGCAACTTG ACGAATCTAA GAGAAATGAC 241 CTTATCCAAA GCTGGCTGGG TCCAGTACAG GAATAGGTTT CGACCGACCC AGGTCATGTC 271 TTCTTATCTC GAGTATTTAC TAATAGCTTG AAGAATAGAG CTCATAAATG ATTATCGAAC 301 GTGTTTGGTA CCAG GACCG TGTCTACGAG CACAAACCAT GGTCACTGGC ACAGATGCTC 331 AAATTAAAGG ATCTGGAAGA AGGCATTCAA TTTAATTTCC TAGACCTTCT TCCGTAAGTT 361 GCATTAATGC GTGAGTTAGA AGATGGAAGC CGTAATTACG CACTCAATCT TCTACCTTCG 391 CCTCGTGCAG GCCAAATCTT GAAACAGACT GGAGCACGTC CGGTTTAGAA CTTTGTCTGA 421 TACGACAAAT TCGACACGAA CCTTCGCTCT ATGCTGTTTA AGCTGTGCTT GGAAGCGAGA 451 GATGATGCGC TGTTGAAGAA C TATGGATTA CTACTACGCG ACAACTTCTT GATACCTAAT 481 CTCTCCTGTT TTAAAAAGGA TCTTCATAAA GAGAGGACAA AATTTTTCCT AGAAGTATTT 511 GCCGAAACAT ATTTGCGTGT AATGAAATGT CGGCTTTGTA TAAACGCACA TTACTTTACA 541 CGTCGCTTCG TTGAGAGCTC CTGCGCTTTT GCAGCGAAGC AACTCTCGAG GACGCGAAAA 4. A bacterium belonging to the genus Bacillus transformed with the recombinant plasmid vector according to claim 1. 5. A method for producing porcine growth hormone, which comprises culturing the Bacillus bacterium according to claim 4 and recovering the produced porcine growth hormone.