JP3346764B2 - New cDNA fragment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel cDNA fragment useful for the production of a bioactive peptide, and more particularly to a peptide having human brain natriuretic activity (human BN).
The present invention relates to a cDNA fragment containing a nucleotide sequence encoding P), a method for producing the same, and mRNA for synthesizing the cDNA fragment.

[0002]

2. Description of the Related Art The structure determination of new natriuretic peptides secreted from rat and human atria has been published one after another in 1983-1984 [for example: Biochem.
iophys. Res. Commun., 117, 859 (1983); Biochem. Bio
phys. Res. Commun., 118, 131-139 (1984)]. These peptides are atrial natriuretic peptides (hereinafter A)
NP), a powerful natriuretic effect,
It has a vascular smooth muscle relaxing action and is attracting attention as a new type of peptide-based cardiovascular drug.

On the other hand, in 1988, a new diuretic peptide was isolated and purified from porcine brain, and its structure was determined. The porcine brain natriuretic peptide (hereinafter referred to as porcine BNP) was obtained.
[Nature, 332, No. 6159, 78-81 (1988);
Biochem. Biophys. Res.Commun., 155, 726-732 (198
8)]. The pharmacological action of brain natriuretic peptide (BNP) is almost the same as that of ANP, and has diuretic action, natriuretic action, blood pressure lowering action, chick rectal relaxation action, etc., and its specific activity is almost equivalent to ANP. However, rectal relaxation activity is 3 to 4 times higher for BNP than for ANP. Due to such properties, BNP is also expected as a new peptide-based cardiovascular drug. For pig BNP,
Studies at the DNA level have also been performed, and cloning of a cDNA having a nucleotide sequence encoding porcine BNP and its precursor has been reported [Biochem. Biophys. Res. Com.
mun., 157, 410 (1988)].

[0004]

Since BNP is used as a therapeutic drug for human diseases, the development of human-derived BNP has been demanded due to problems such as antigenicity. However, the existence and structure of human-derived BNP at the protein and peptide levels and at the DNA level have not yet been proven.

[0005]

Under such circumstances, the present inventors have conducted various studies to obtain human-derived BNP (human BNP). As a result, a cDNA fragment encoding a porcine BNP precursor was used as a probe. By screening a human tissue cDNA library, cDNA encoding BNP can be extracted from the human tissue.
Was successfully cloned, and the present invention was completed.

That is, the present invention relates to the following amino acid sequence :
Encoding a human brain natriuretic peptide consisting of
It is intended to provide a DN A.

[0007] Ser Pro Lys Met Val Gln Gly Ser Gly Cy
s Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Le
u Arg Arg His

[0008] The present invention also is to provide a manufacturing method of the cDNA A. The present invention also is intended to provide a mRNA for the synthesis of the cDNA A.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The cDNA fragment of the present invention is prepared, for example, as follows.

First, total RNA was isolated from human tissues thought to contain human BNP.
A is purified and a cDNA library is constructed by a conventional method. Subsequently, a human BNP clone is screened from this cDNA library by hybridization using a cDNA fragment encoding a porcine BNP precursor as a probe, to obtain a cDNA fragment of the present invention. Hereinafter, a method for producing the cDNA fragment of the present invention will be described.

(1) Construction of cDNA Library Human tissues for preparing mRNA include human brain,
A human atrium or the like is used. RNA can be isolated, for example, by homogenizing a human atrial fragment with guanidyl thiocyanate and performing cesium trifluoroacetate equilibrium density gradient ultracentrifugation. mRNA was purified using oligo (d
T) Performed by a conventional method using cellulose column chromatography. To synthesize cDNA from the obtained mRNA, for example, a method using a cDNA synthesis kit (manufactured by Pharmacia), the method of Okayama-Berg, the method of Grabler and Hoffman or a modified method thereof, and other commercially available kits are used. It is carried out by a conventional method. After adding a restriction enzyme EcoRI adapter to the obtained cDNA, the 5 ′ end is phosphorylated with T4 polynucleotide kinase or the like, and the vector (for example, λ
gt10), followed by in vitro packaging to construct a cDNA library.

(2) Screening of human BNP clones Screening of human BNP clones is carried out using a probe of a porcine BNP cDNA fragment as a probe. This cDNA fragment was cloned from cDNA of porcine BNP [Biochem. Biophys. Res. Commun., 157, 410 (1
988)], pB which is a clone of incomplete length obtained in the process of
NP-82 (320 bp) was replaced with restriction enzymes Xho I and Rs
a I digested 120 bp fragment (porcine BNP)
Porcine BNP-26 (comprising 26 amino acids) and its upstream 30 bp DNA fragment), and a probe is prepared by labeling this cDNA fragment with ³² P. The probe is hybridized with the aforementioned cDNA library, and a positive clone is selected. The cDNA fragment of the present invention can be obtained by digesting the selected positive clone λhBNP-57 with a restriction enzyme.

The nucleotide sequence of the obtained cDNA fragment can be determined by a conventional method, for example, by incorporating the cDNA fragment into a sequencing vector to prepare phBNP-57, and then preparing a restriction enzyme map of the cDNA region. D obtained using a restriction enzyme that cuts to length
Each of the NA fragments is re-incorporated into a sequencing vector and subcloned, and the entire nucleotide sequence can be determined by the method of Sanger et al.

The nucleotide sequence of the cDNA fragment of the present invention thus determined and the amino acid sequence containing human BNP are as shown in FIG. In the base sequence of FIG. 2, 1 to 402 are human B consisting of 134 amino acids.
Encodes an NP pre-precursor polypeptide, of which 79
~ 402 is thought to encode a human BNP precursor polypeptide consisting of 108 amino acids. This indicates that the structure of the signal peptide in front of the precursor is very similar between pig and human, and that Arg-Ser-His-Pro-Leu-
The amino acid sequence of Gly (corresponding to base numbers 73-90) is also present in porcine BNP, in which the Ser-His bond is broken and a porcine BNP precursor is produced [Biochem. Biophy.
s. Res. Commun., 157, 410 (1988)]. Of these, 307 to 402 are thought to encode human BNP-32 consisting of 32 amino acids.

This cannot be determined because human BNP has not yet been isolated as a peptide, but it is considered that human BNP is produced by processing from the precursor. In the case of pig, pig BNP-26 (comprising 26 amino acids) [Nature, 332, 78-81 (1988)] pig BNP-
32 (consisting of 32 amino acids) [Biochem. Biophy
s.Res.Commun., 155, 726-732 (1988)),
In humans, ANP (amino acid 28)
Residue) [Biochem. Biophys. Res. Commun., 11
8, 131-139 (1984)] In rats, rat αANP (consisting of 28 amino acids) [Biochem. Biophys. Res.
mun., 117, 859-865 (1983)]. These have all been cleaved and formed after Arg and Pro-Arg present in their precursors. In the case of human BNP, Arg existing before a peptide consisting of 23 amino acid residues from Cys (at position 112) to His (at position 134), which is considered necessary for activity
Is the 102nd Arg and has the structure of Pro-Arg, so it was thought that it would be processed later to produce human BNP-32 consisting of 32 amino acids.

In the case of ANP, it is known that the precursor also has a physiological activity, and it is considered that the case of human BNP is the same. When considered as a drug, the precursor, human BNP-32
Both are useful substances.

Although the full-length cDNA fragment and the corresponding amino acid sequences are as shown in FIG. 2, only the full-length peptide does not necessarily show human BNP activity. That is, only the full-length peptide produced by this full-length cDNA fragment is not always useful. For example, a peptide having a shortened C-terminal may be obtained.
Alternatively, a peptide exhibiting human BNP activity can be produced by partially substituting codons encoding amino acids. In addition, D encoding these amino acid sequences
It is common sense that the NA sequence is not limited to one type, and once a full-length cDNA fragment is specified, various variant DNAs can be used.
Can be used to produce a peptide having human BNP activity. Therefore, the human-derived brain natriuretic peptide (human BNP) as referred to in the present invention is not limited to full-length human BNP but is a peptide having BNP activity, and is a base encoding human-derived brain natriuretic peptide. The sequence means a base sequence encoding them.

[0018]

The present invention produces human BNP, human BNP precursor, and various peptides having the same biological activity as the cDNA fragment of the present invention by introducing and expressing an expression vector by a conventional method. Can be. The resulting human BNP has an excellent smooth muscle relaxing action,
It has diuretic and natriuretic effects, blood pressure lowering effects, and is highly safe because it is of human origin, such as cardiac edema, renal edema, hepatic edema, pulmonary edema, hypertension, congestive heart failure, acute and chronic It is useful as a remedy for renal failure and the like.

The administration method can be the method used for the administration of peptide drugs, that is, intravenous injection, intramuscular injection, subcutaneous injection, or sublingual administration, intranasal administration, or rectal administration. . The amount of the peptide that can be administered without causing any dangerous or detrimental side effects is preferably in the range of 0.5 μg / kg to 100 mg / kg.

[0020]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Construction of cDNA Library After 3 g of human atrial fragment was crushed with liquid nitrogen, the method of Chirgwin et al. [Chirgwin, JM et al .: Biochemistry, 18, 5294-
5299 (1979)] and an aqueous solution of guanidium thiocyanate was added and homogenized. The total RNA was then separated by cesium trifluoroacetate equilibrium density gradient ultracentrifugation. Then, this was purified by an oligo (dT) cellulose column chromatography by a conventional method, and 37 μg of poly (A) ⁺ RNA (mRNA) was isolated. Then cDN
3 μg of mR using A synthesis kit (Pharmacia)
A cDNA was synthesized from NA, and after adding an EcoRI adapter, the 5 ′ end was phosphorylated with T4 polynucleotide kinase and ligated with the vector λgt10. The vector used was λg digested with EcoRI and dephosphorylated.
t10 arms were used. This λgt10 Arms 2μ
The cDNA used for ligation is equivalent to 0.1 μg of RNA used for cDNA synthesis with respect to g. After ligation, packaging was performed using a packaging kit (Gigapack Gold: manufactured by Stratagene) to complete the construction of the cDNA library. To examine the construction results of the cDNA library, a small amount was used for E. coli c600.
When c600hf1 was inoculated, 90% of the plaques appearing at c600 were clear and 10% were turbid. This indicated that the recombinant phage accounted for 90% of the library. Also, cDNA
The entire library was found to produce about 7 × 10 ⁷ Braaks.

(2) Screening of Human BNP Clone Screening was performed on a 5 × 10 ⁵ plaque that emerged by inoculating a part of the cDNA library into Escherichia coli c600hf1. On the other hand, as a probe for screening, a cDNA fragment of porcine BNP labeled with ³² P was used. This cDNA fragment was cloned from porcine BNP cDNA (Biochem. Biophys. Res. Commun., 157, 4).
10 (1988)].
A 120 bp fragment (porcine BNP) cut out from BNP-82 (320 bp) with restriction enzymes Xho I and Rsa I
Porcine BNP-26 (consisting of 26 amino acids) and its upstream 30 bp DNA fragment) were purified and prepared by acrylamide gel electrophoresis. First, the plaque was transferred to a nylon filter, neutralized after alkali treatment, and the DNA was fixed by ultraviolet irradiation. 5
4 × SSC containing Denhardts solution, 100 μg / mL denatured salmon sperm DNA and 0.1% SDS
The filter was immersed in a solution of 0.6 M NaCl and 0.06 M trisodium citrate and prehybridized at 60 ° C. for 3 hours. Subsequently, a probe labeled with ³² P was added to the hybridization solution (the same composition as the prehybridization solution) at a concentration of 2 × 10 ⁶ cpm / mL using a Randomprimed DNA labeling kit (manufactured by Boehringer Mannheim). Was incubated at 60 ° C. overnight. Next, the filter was washed with a 2 × SCC solution containing 0.1% SDS, air-dried, and then subjected to autoradiography. This gives 55
Hybridization positive plaques were obtained. An attempt was made to determine whether or not these 55 positive plaques hybridized using DNA (680 bp) encoding human ANP as a probe. As a result, it is clear that this cDNA is different from a known cDNA encoding human ANP. 5 above
Five positive plaques were monoclonally cloned, and then λ phage DNA was prepared by a conventional method. When a DNA fragment obtained by digesting this with the restriction enzyme EcoRI was examined, it was found that a clone named λhBNP-57 had a maximum length of about 7 μm.
A 00 bp cDNA was inserted. The insert cDNA was incorporated into Blue Script (KS (+)), a vector for sequencing, manufactured by Stratagene to prepare bhBNP-57. E. coli containing this plasmid is E. coli. coli HB101 / phBN
It was named P-57 and deposited at the Japan Fine Industrial Research Institute as Fine Industrial Research Institute Deposit No. 2299 (FERMBP-2299). Next, a restriction map of this cDNA region was prepared. Further, using a restriction enzyme cleaved to an appropriate length to obtain a cDNA fragment,
Each was reassembled into Blue Script and subcloned. FIG. 1 shows the restriction enzyme cleavage site of the inserted cDNA region and the strategy for determining the nucleotide sequence. The nucleotide sequence was determined by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA, 74, 54).
63-5467 (1977)). This cD is shown in FIG.
1 shows the nucleotide sequence of NA and the amino acid sequence corresponding to the nucleotide sequence. The sequence of the inserted DNA has a long translation region starting from the translation initiation codon ATG and ending at the translation termination codon TAA. This cDNA having a total length of 692 bp is a 5 'untranslated region up to base pairs -99 to -1;
8 encodes a signal peptide and 79-402 are thought to encode human BNP precursors. Of these, 307 to 402 are human BNP-32 (amino acid 32).
(Consisting of residues). Also 4
03 to 593 are 3 'untranslated regions. Among them, the amino acid sequence corresponding to the nucleotide sequence of 307 to 402 encoding human BNP-32 is amino acid 17 of which
It has a cyclic structure in which residues are formed by cysteine disulfide bonds, and has a structure very similar to that of porcine BNP.

Example 2 Production of Polypeptide A cDNA excised from λhBNP-57 with the restriction enzyme EcoRI was inserted into a plasmid Bluescript to utilize a recombinant plasmid, clone phBNP-57, to obtain γ-BNP-57. A vector that produces BNP can be obtained. That is, phBNP
Immediately before the γ-BNP coding region of the -57 inserted cDNA, a new restriction enzyme recognition site and a translation initiation codon (ATG) are introduced by site-directed mutagenesis, and the fragment is separated using this new recognition site. . Next, the above fragment is inserted immediately downstream of the promoter of the plasmid for the expression vector, and this plasmid is introduced into Escherichia coli. The E. coli is cultured with sufficient nutrients to synthesize the polypeptide, and then γ-BNP is recovered. Further, by changing the position and base sequence of the position-directed mutation on the inserted cDNA of phBNP-57,
A fragment for producing NP-32 or BNP in Escherichia coli is obtained, and an expression vector into which these fragments have been inserted is introduced into Escherichia coli, and BNP-32 or BNP can be recovered by culturing the fragment. .

[0024]

[Sequence List] SEQUENCE LISTING <110> Shionogi & Co., LTD.MATSUO, Hisayuki <120> Novel cDNA Fragment <130> P05521212 <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 32 <212 > PRT <213> Homo sapiens <400> 1 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30

<210> 2 <211> 102 <212> DNA <213> Homo sapiens <400> 2 ccacgaagcc ccaagatggt gcaagggtct ggctgctttg ggaggaagat ggaccggatc 60 agctcctcca gtggcctggg ctgcaaagtg ctgaggcggc at 102

[Brief description of the drawings]

FIG. 1 shows a strategy for determining the nucleotide sequence of a cDNA fragment inserted into a positive clone λhBNP-57 and a restriction map of the cDNA fragment.

FIG. 2 c determined according to the strategy of FIG.
The base sequence of the DNA fragment and the amino acid sequence corresponding to this base sequence are shown.

Continuing from the front page (72) Inventor Keiji Maekawa 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo Inside the Daiichi Pharmaceutical Central Research Laboratory (72) Inventor Naoto Minamino 5618 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki-gun, Miyazaki (72) Invention Person Kenji Samukawa 1520-24 (72) Inventor Toshiyuki Matsuo 653 Kihara Kiyotake-cho, Kiyotake-cho, Miyazaki-gun, Miyazaki Prefecture JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/19 C07K 14/47 BIOSIS (DIALOG) GenBank / EMBL / DDBJ / GeneSeq PubMed

Claims (10)

(57) [Claims] 1. A cDNA encoding a human brain natriuretic peptide having the following amino acid sequence: Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 2. The c according to claim 1, which comprises the following nucleotide sequence:
DNA. ccacgaagcc ccaagatggt gcaagggtct ggctgctttg ggaggaagat ggaccggatc agctcctcca gtggcctggg ctgcaaagtg ctgaggcggc at 3. The cDNA according to claim 1, which is obtained from mRNA purified from a human tissue containing a human brain natriuretic peptide. 4. cDN constructed from mRNA purified from human tissue containing human brain natriuretic peptide
The cDNA according to any one of claims 1 to 3, which is obtained from the A library. 5. The method according to claim 1, wherein the human tissue is a brain or an atrium.
The cDNA according to any one of claims 1 to 4. 6. The amino acid sequence according to claim 1 , which is purified from a tissue containing a human brain natriuretic peptide.
MRNA encoding . 7. The mRNA according to claim 6, which is purified from total RNA isolated from a human tissue containing a human brain natriuretic peptide. 8. The method according to claim 6, wherein the human tissue is a brain or an atrium.
Or the mRNA of 7; 9. The mR according to any one of claims 6 to 8,
6. A cDNA library is constructed using NA, and then a human brain natriuretic peptide clone is screened for the cDNA library by hybridization using a probe. Method for producing cDNA. 10. The cDNA according to claim 9, wherein the probe is a DNA fragment encoding porcine BNP precursor.
Manufacturing method.