JPS60501562A - Human calcitonin-related peptides and pharmaceutical compositions - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to peptides, pharmaceutical compositions, methods for producing peptides, and genes encoding peptides. offspring, a vector containing this gene and a host organism transformed with this vector, and and gene probes and antibodies used for diagnosis.

Background of the invention The calcitonin gene system has been the subject of considerable research in recent years. In particular, rat karsi Tonin gene expression research has demonstrated that various peptides can be extracted from a single gene. apparently tissue-specific selective mRNA species to produce hormones. (Craig, Earl, Kay et al. (1983), Genetic Engineering (Genetic~244). Each mRNA species is Post-translational processing results in polyclonal white matter encoding and rat Calcitonin or rat calcitonin gene related Vepti 1 (Ran) CGRP ) is produced. ) CGRP is widely used in discontinuous areas of the central nervous system and peripheral nervous system. This pending iodine with potential biological activity is known to be distributed in Loppa patent application BP-AI-0070186 and BP-A1-00706 No. 75 (R, K, Flave and I, Matsukin type) K is human calcito of the carboxy-terminal peptide (katacalcin) designated as nin and PDN-21. The manufacturing method is described. Human calcitonin and katacalcin are human calcito produced as a polypeptide encoded by Nin mRNA (Flaib et al. 1982) The present inventors have demonstrated that human calcitocellular carcinoma is transcribed into human medullary carcinoma cells and Discovery of the human calcitonin gene region located at the end of the 3' translated region of the nin mRNA did. This region includes transcriptional intron regions, splice junctions, and previously unknown human peptides. The open reading frame encodes the sequence and ends in the region of IJA residues. Consists of 6' untranslated region. One of these peptides is apparently pine) similar to CGRP This will be referred to as human calcitonin gene-related peptide (human CGRP). It is called.

Summary of the invention According to a first feature of the present invention, a human calcitonin gene-related peptide is provided.

According to a second feature of the invention, the structure: Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr -Hi e-Ar g--Leu-Ala-Gly-Leu-Leu-8er- Arg-8er-Gly-Gly--Va'1-Val-Lys-ASn-As n-Phe-Val-Pro-Thr-Asn--Val-Gly-8er-L ys-Ala-Phe-amide (1).

This novel peptide is a protein cleavage agent that recognizes basic amino acid residues located on its flanks. As part of polyproteins that are specifically cleaved in vivo within the secretory pathway by enzymes It is formed. This peptide (Tatemoto and Matt (1981) PNAS 7 8, 6'603-6607 and was described as PAF-37) It was found to have potential biological effects in regulating vascular function. In particular, this peptide was found to induce a decrease in blood pressure and increase 6-temperature and power.

In the second feature of the present invention, the peptide is used as a medicine, preferably for the treatment of hypertension. provided for medical use. Primary cardiac surgery (e.g. open heart bypass surgery) In the post-surgical phase, patients respond to the stress of surgery through extensive vasoconstriction. It is normal to do so. This has the effect of raising the patient's blood pressure, which puts stress on the heart. Make it tense. Vepti-P of the present invention acts as an antihypertensive agent and can increase the power of puerperium. I understand. These linking effects have potential applications in post-surgical treatment. many People suffer from hypertension, which is a contributing factor to the high incidence of heart disease. ing. This peptide can be used for hypertension management.

In a third feature of the invention, a peptide of structure (1) and a pharmaceutically acceptable Provide a pharmaceutical composition comprising excipients. The composition may be injectable. Pharmaceutical group The composition can be contained in the body in a slow release composition or peptide system; Or it can form part of it. Such slow-release drugs can prevent long-term hypertension. Hello used for management.

Further, the present invention provides a method for treating hypertension, comprising administering an effective amount of a peptide having structure (1). Ru. Additionally, a sixth feature of the present invention provides that the peptide of structure (I) is pharmaceutically acceptable. A method of manufacturing a pharmaceutical composition is provided.

The peptide of structure (1) can be produced by various methods.

This peptide is produced by chemical peptide synthesis using techniques known in the art. can be done. Purification of peptides using ionic, reversed-phase chromatography techniques can be done.

In another feature of the invention, at least the amino acid sequence of the formula Ala-Cyq-Asp-Tbr-Ala-Thr-Cys-Val-Thr- His--Arg-L　eu-A　], ]a-Gly-Leu-Leu-8er Ar　g-8er　-G　y-(wherein, R' is -H or an amino acid residue) or a peptide that can be converted into an amide in vivo or in vitro. provide R' is -017-1-Gly-Arg-Arg-Arg-Arg or -Gly-Lys-Lys-Arg is acceptable, but -017 is preferable. Carboxy The terminal residue R' is the amino acid residue of the adjacent phenylalanine amino acid residue in vivo or in vitro. It can be enzymatically converted into rice. When R' is -GlyO, an enzyme suitable for this conversion is is yeast carboxypeptidase Y or mammalian amidating enzyme (Bloodbase). Lee, A, F, Queenie. M, D, A and Smith, D, G ( 1982) Nature, 298, 686-6'88 ).

Alternatively and preferably, the peptide is produced by recombinant DNN production. .

As a fourth feature of the present invention, the intermediate peptide is a peptide according to the fourth feature of the present invention. A host organism transformed with a vector containing a gene encoding an intermediate peptide The intermediate peptide is obtained by culturing the structure (1 ) provides a method for producing a peptide.

The intermediate peptide contains at least one of the proteins produced in the transformed host organism. A fusion protein containing the amino acid sequence described in part and above (Ill) is good.

Desirably, the fusion protein is produced at high levels by the transformed host organism. Contains protein. Suitable proteins are at least partially chloramphenicol. Cetyltransferase (cAT) protein or at least a portion of β-galactin Contains tositase protein white matter. The fusion protein is present at high levels in the transformed host organism. The amino acid sequence described in (■) above is bonded to the carboxy terminal group of the produced protein. peptides having a sequence.

Peptides can be linked to proteins via selective chemical or enzymatically degradable bonds. good. This bond may be a methionine or glutamic acid residue. Methionine is a bromide Glutamic acid can be selectively cleaved by ammonium, and glutamic acid is an acid protein derived from turgium. rotease (Ec3..4°23.14), sea urchin protease (Kc3.4.2 4°12) or preferably Staphylococcal protease (EC3,4,21) , 19) (H) The C0RP sequence can be selectively cleaved using Met or does not contain Glu residues). The bonded lysine-arginine peptide has two functional groups. stomach. Cleavage of this bond is performed using mouse submandibular gland protease or preferably clostripan. This can be done using (EC3°4.21.6.).

The peptides according to the first and second features of the present invention are produced as polyproteins in the body. Ru. This polyprotein white matter is processed in the body, and the amide of the first or second feature of the present invention is The amine and carboxy end groups are cleaved leaving the amino acid pezotide. Main departure The sixth feature of Ming is at least the following amino acid sequence.

Arg-Echoe-Ala-Gln-Lys-Arg-Ala-Cys -Asp--Thr-Ala-Thr-Cys-Val-Thr-His-Ar g-Leu-Ala--Gly-Leu-Leu=Ser-Arg-8er41 y=Gly-Val-■a1. Gene encoding intermediate peptide containing -Lys- The intermediate peptide is obtained by culturing a eukaryotic host organism transformed with the vector containing the progeny. , this intermediate peptide is processed by the host organism, and then the peptide is A method for producing Bezochi 1 having structure (1) is provided. Eukaryotic host organisms are Protein degradation capable of producing peptides of structure I from polyproteins containing the amino acid sequence ■ A tissue-cultured clear mammalian cell line that can contain enzymes and amylating enzymes is desirable. Desirably the intermediate peptide is produced as a fusion protein.

In the processing of polyproteins, tetrapeptides are produced. This te The tiger peptide also has biological activity and is a feature of the present invention, and has the following sequence: Asp- It has Leu-Gln-Ala (■). This peptide is designated as 'PDA-4' and was found to have biological activity in regulating cardiovascular function. Especially this pep Tide was found to induce a decrease in blood pressure and increase heart rate.

An eighth feature of the present invention is that the peptide having the sequence (■) is used as a medicine, preferably for hypertension. It is used for the treatment of.

A ninth feature of the present invention is that the peptide having the sequence ffV) and a pharmaceutically acceptable excipient It is to provide a pharmaceutical composition comprising an excipient. The power of this composition to be injectable 1 Okay.

The pharmaceutical composition may be a slow-release type composition in the living body or may be contained within the Muma peptide system. ℃ can also form part of it. The composition comprises a peptide of structure I. It may contain a combination of a peptide having the sequence ① and a peptide having the sequence ① and a pharmaceutically acceptable excipient.

The ninth feature of the present invention is that the gene encoding PAF-57 (structure I), F-37-R' (Structure ■), PDA-4 (Structure PJ) or [Non-pro Provides a "set sink" pen (structure ■). Preferably, the following Mino acids, lower half of Figure 2 1) 1 to 37, 1i) -3 to -7, 1ii) +6 to +9 or iv) “Identification described by nucleotides corresponding to amino acids 7 to +9” Provide the gene. Furthermore, the present invention provides a method for nucleotides 1 to 1256 substantially shown in FIG. Provide the nucleotide sequence. Furthermore, the present invention provides vectors containing any of the genes of Kowara. A vector and a host organism transformed with the vector are provided. suitable host organism The body contains bacteria (e.g. Escherichia coli) and yeast (e.g. Saccharomyces cerevisiae). d) and tissue cultured mammalian cells.

H) QGRP production is a selective mediating tissue, a tissue that relies on mRNA processing. There is (Nidoprusk, M, R, etc., Nature) (1 984) - raised).

In certain cells, such as those found in medullary thyroid carcinoma and lung cancer, C0RP is Produced in Bell. Therefore, the presence of human CGRP can be diagnostic of abnormal tissue. Ru. Furthermore, the present invention is used for testing C0RP gene expression and abnormal gene tissue. Provide diagnostic reagents (including antibodies and gene probes).

As an eleventh feature of the invention, the invention provides a peptide of structure I or an antigenic determinant. (with a detectable label). This label is based on enzymes and color development. It may be a fluorophore, a fluorophore, or a chemiluminescent group. However, the most desirable is radioactive An isotope, for example, at the histidine amino acid residue at position 5 of the amino acid sequence of a peptide. 1251 to join. The labeled peptide is a peptide of structure I or 125 It is preferable to include a portion thereof containing an optionally labeled tyrodinoamino acid residue. Pep The tido moiety is amino acid 25 to 37 (amidated phenylalanine) or amino acid 1 to 8 is desirable.

The twelfth feature of the present invention is that the peptide of structure (1) has specificity for antigenic determinants. The objective is to provide antibodies that This antibody can be polyclonal or monoclonal But that's fine. Antibodies can be labeled with a detectable label.

The reagents according to the eleventh and twelfth features of the present invention can be used in immunoassays, preferably radioimmunoassays. Immunocytochemical analysis of immunoassays, peptides of structure I in samples, or tissue parts. A specific one is fine.

mRNA A encoding PAF-37 or a nucleotide sequence containing that mRNA It is also possible to test for columns.

Therefore, as the eleventh feature of the present invention, the boxes 1 to 1256 shown in FIG. A DNA sequence containing more than 15 types of nucleotide sequences selected from The first step is to provide a blit probe. Probe (also can be immobilized on solid phase) can be labeled with a detectable label.

Such probes can be used to determine genetic organization using DNA + RNA plotting technology. or gene expression, and/or determine the normal location (in It can be used to confirm with hybrid cells of 5 ita).

The desirable blow play of the sixteenth feature of the present invention is shown in FIG. It has a sequence selected from the following. This desirable type of probe Q'1. Hi To test for human) CGRP as opposed to tocalcitonin gene organization or expression. You can use it. In particular, this probe is compatible with human CGRP mR11A and human C() RP structural gene.

Another desirable feature of the present invention is the 2°C probe (1.726 shown in Figure 2). The sequence consists of ~1256 nucleotide sequences. This desired temperature and type ) Can be used to test organisms for the 0-rope G-mahitocalcitonin C0RP gene. I can do it. In particular, this probe is located in the intron region of the QGRP structural gene. I'm going to have a good time in the area.

The invention will be explained by the following description with reference to the following drawings.

Figure 1 shows recombinant cilasmids phT13'', phTB t5 and phTB. FIG. 58 is a schematic diagram of overlapping cDNA sequences cloned in 58.

Figure 2 shows recombinant plasmids phTB3, phTB(5 and phTB58). The complete nucleotide sequence derived from

Figure 6 shows cilasmids pcT201, pCT202, pcT203 and pCA FIG. 2 is a schematic diagram of the structure of T-CGRP.

Chloramphenicol acetyltransfera in extract of HB 1Q 1 Polyacrylic acid showing the presence of fusion protein of CGRP (CAT) and CGRP It's Midel.

Figure 5 shows protein extract of E. coli HB1Qi transformed with pCAT-C0RP. ) (!Results of radioimmunoassay showing the presence of GRP antigenicity determinants) This is a graph that proves the result.

Figure 6 shows drug sensitivity curves of 6-stroke frequency and mean arterial pressure in rats (·=food Salt water, ○=hi) cGRP.

△ = Lalan cGRP, Ronihito C0RP + Propranolol, -bihuman CORP + mevilamine + cimetidine).

Figure 7 shows the 6 degrees of PDA-4 (and human C0RP) intravenously injected into rats. Tachograph trace demonstrating the effect on mean arterial pressure.

FIG. 8 shows drug response curves of six pressures and force in guinea pigs.

Figure 9 shows medullary thyroid cancer tissue and plasma extract of a patient with medullary thyroid carcinoma. Radioimmunoas demonstrating the presence of GRP and production of Ccup by lung cancer cell lines. This is a graph proving the assay results.

110 shows the relationship between calcitonin in medullary thyroid carcinoma and lung carcinoma cell lines and in humans. [:! GRP RNA O) Shows RNA plots demonstrating various expressions.

Figure 11 shows the DNA of human placental tissue and medullary thyroid carcinoma tissue or lymphocytes from a single patient. When comparing DNA, we prove that various organisms have homologous genes to the human CGRP gene A DNA plot is shown.

Pending European Patent Application No. EP-Ai-0070675 describes human bone marrow Whole cells isolated from quality thyroid cancer tissue! Using J(A)-containing RNA, 5CD Composition of NA library (Biochemistry managers such as Allison and J.A.) (BiochemJ, ) (1981) 199 725-731) and from this library =, ph’r = B3 and phT-B6 Human calcitonin mRNA6 cloned in two separate plasmids Nucleotide sequence analysis of most of the Nature) (1982) 295.645-347) is described4. There is. During this study, one recombinant containing an inserted cDNA fragment of approximately 1600 bp Plasmid phT-B53 was identified through preliminary screening; No analyzes of 4 or higher were performed.

Because it is a very large molecule, it was not thought that it would show calcitonin mRNA. . The cDNA inserted into this cilasmid was subjected to subsequent restriction enzyme analysis (see Figure 1). For example, the common position is shown in pHTB3 and further cloned within ph'r-853. The cDNA represents the sequence downstream from the pre-established sequence, and represents human calcitonin mRNA. It was noted that the complete 3' untranslated region of A. Figure 1 shows the sequence specificity Restriction sites and calcitonins that separate regions of the sequence used as lit probes The relative positions of C0RP, CORP and the common amine-terminated peptide are shown. The vertical dashed line is The Pst1 site separating cDNA and plasmid sequences is shown. phT-B 5 Nucleotide sequence analysis of the entire cDnA sequence inserted in 8'17c was performed using the method previously described. (Flave R K, Hall L, Nidprtsk M R, Aυ Sun, J. E. and Matsukintyre, I. (1982), Nature. re) 295.345-347) - It was carried out using hd shown in Fig. 2. The numbers are Known nucleotide sequences of rat calcitonin and CGRA RNA transcription Compare with Gyatsude, which was introduced to maximize homology. human nucleotide sequence The number immediately above is the nucleus from the poly(A) tail cloned into pHT B53. Shows the relative slowness of ocide. The number on the human protein sequence is calcitonin or QGRP. means the position of the amino acid relative to Another or The new amino acid or nucleotide is shown immediately below the column in question. potential Boriadenylation symbols are underlined. Arrow (↑) is mature calcitonin It means the 6' end of mRNA, and the (Mu) mark indicates an additional int in the human sequence. Pointing to possible positions for Ron. The dashed line is the region of the human sequence without the rat calcitonin gene. The inserted cDNA sequence contains 1615 bp and extends from the 6' end. It ends in the region of the IJ (A) residue. Of these, the first 356 from the 5' end bp is the same as above, human calcitonin mR encoding katacalcin A portion of NA and the AATAAA box and a port in mature calcitonin mRNA. The 6' untranslated region containing the 12 nucleotides shown preceding the tail (A) Shown the whole thing. Analysis of the remaining sequence encodes 53 amino acids, followed by a terminal codon. It was shown to contain a single open reading frame.

This is the striped rice junction receptor site (C) NAG/G (Mount Varnish M, Nucl θic Ac1d Res, ) (1982) 1 mouth 450-463), it is located just before the 5' side, and is The 645 nucleotides of the ``Intron'' uniform sequence are known by 6 adenosine residues. isolated from calcitonin mRNA sequence.

However, the only recognizable donor splice junctions are intron-uniform sequences and human calcitosynthesis. It did not exist among the sequences known to exist. new The standard open reading frame is a region of 451 nucleotides containing two boriadenyl symbols. , the first (AATAA) is 26 bases downstream of the terminal codon and the second (AATAA) is AAAATTAAAAAA) is located 18 nucleotides before the terminal poly(A) region. Ta. The coding sequence consists of a pair of basic amino acids (-1, -2), plus five amino acids (-6 to -7) provides an amine terminus and a glycine residue (+1), four basic amines. Carboxyl with amino acids (+2 to +5) and tetrapeptides (+6 to +9) It contained human C0RP (a 37 amino acid peptide) flanking the end. Group Presence of lysine reflects in vivo requirement for amidated carboxyl-terminated phenylalanine Shiku Platoberry, A.F. et al., Nature (1982) )-298, 686-688), a total of 3786 for amidated human cGRP. He became Mr. Comparing the predicted human amino acid sequence with the rat sequence (Amara et al. The results showed sequence conservation, with 7 amino acids changing from 53 amino acids. , 4 are Hi-CGRP (alanine 1, aspartic acid 6, asparagine 25 , lysine 35), and the remaining six (-7, -6, -5) are 5-amino acid amines. Located in the terminal leader sequence. Of the latter, the first amino acid (arginine-7) Based on the location of splice junctions and the rat calcitonin gene, the inventors Designated by homology with child. Significant sequence conservation occurs at the nucleotide level within the coding region. It is clear that the human sequence and the available rat CGR,P mRNA sequence A comparison shows that it is significantly reduced in the 3' non-coding region. Human CGRP amino acid sequence and others Compare the protein sequences of (Wilbur, F.G. et al., PNAS (198 3) 80°726-730) and other known peptides including calcitonin. It was shown that there is no significant homology (with the exception of rat C0RP). At most, nine Comparable amino acids identified by alignment of human C0RP with salmon calcitonin It was done.

This study was conducted using cDNA fragments isolated from phT-B 3 and phT-B 5 F3. The inventor discovered that the phT-858K cloned cDNA was partially derived from furosetucin. Restricted human categorization of DNA showing restricted boriadenylated RNA transcription. Established by the method. In addition to the “introns” identified by nucleotide sequence analysis, In addition, one intron was mapped to the 6' side of the cGRP coding sequence, and the others were mapped to the 6' side of the cGRP coding sequence. The r-tum structure closely resembles that of the calcitonin gene (genomicorgan mapping to calcitonin 1 sequence 5'llll suggesting (Rosenfelt M.G., Marmod G.I., Amara N.I. S.C., 7 Sutunnn el tahru, Sochenko, hi, -.

E., Rivière G.E., Vert W. Duffreu Oyohie Evans. R.M. (1983), Nature, 604.129 ~135).

However, the calcitonin exon and C! "Intron" that separates the GRP coding sequence The similar sequence actually shows a Ke8tum structure, and calcitonin isolated from phT-853, 1125 bp Sph I/Pvu including intron and C0RP sequence The l'l DNA fragment (see Figure 1) has "intron" specificity... As determined by the hybrid against the probe, K, Sph l / Pvu ■ Electrophoresis with Ketum fragments of the same size after Zapon method analysis of restricted human placental DNA. move.

Humans by recombinant DNA technology (:! Production of GRP Humans by recombinant DNA technology) To produce cGRP, chloramphenicol acetyltransferase produces a fusion protein consisting of the active portion of proteinaceous protein (cAT) and the desired intermediate peptide. We created vectors that can occur. (This vector is part of the pending international patent application PCIT) /t) B 84100179, UK Patent Application No. 8413601, 198 (Disclosed in the application filed May 24, 2013).

The plasmid is a chloramphenicol weakly resistant R1 OR-plasmid mutant. Pst1 digested the body DNA, and then the single-Pst1 fragment was transferred to the plasmid pBR filter. 22 (Iita et al. (i932) FMBO J, 1.755-759). Cilasmid, pBR322:Cn104 obtained and deleted The cAT enzyme with the last seven amino acid residues at the carboxy terminus removed by Code the element. This deletion is due to a naturally occurring in vivo mutation involving insertional element S1. According to However, the generated DNA molecule has a terminal colloid at the end of the CAT engineering structural gene. Does not have a dong.

Therefore, the liposome translates into protein and R NA was transcribed from engineered SI DNA. The net result is a longer CAT than the natural enzyme. □Protein has 19 amino acid residues, and the last 26 amino acid residues are engineered SiD directed by the NA sequence. This structural gene also creates the desired fusion protein. Lacking suitable restriction sites useful for this purpose, a series of DNA manipulations were performed.

Plasmid I)B containing the PSt1 restriction fragment containing the above mutation c AT I gene R322: isolated from Cn 104 and dephosphorylated from plasmid pAT 153 It was bound to the Pst1 site. In this direction, CATI and β-lactamase protein Since both motors transcribe in the same direction, plasmid pAT/Cn10 4b (Figure 6) was selected. This cloning method is unique Tth 111 ■It was mainly composed of plasmids with restriction sites. , this disconnection The site was linked to the end of the CAT1 structural gene], derived from SI DNA, and There are 19 amino acid codons in the 26 amino acid residue extension shown below.

Cilasmid pAT/Cn 104 b is Tth 111. Straighten with I , digested with BAL 31 exonuclease. Take samples at a series of time points, The reaction was stopped using excess EDTA. BAL 31 Any abnormality caused by digestion Slippery ends were also filled using the Flenow fragment of DNA polymerase I. Then these plasmid DNA molecules were dephosphorylated using calf intestinal phosphatase.

Next, the sequence 5' -TCA GATCTGGAGCTCCAGATCTGA - Kinase-treated linker-1R14'O with 3' was added to each plasmid time point sample ( Plasmid time point Sample). After connection , the plasmid DNA was digested with Sstl restriction endonuclease and religated. , only one linker was present in each cilasmid.

These sets of DNA molecules were transformed into E. coli DH1, and the fusion vector plasmid was transformed into E. coli DH1. 20 tt, Q/rrre noticeable in Noshiichi agar containing chloramphenicol It was selected on the basis that it grows in

A small scale plasmid preparation was made. Single 5iit 1 restriction site (linker-D (derived from NA) and simultaneously digested with EcoRI and Bgl 11. A number of plasmids were isolated that yielded small DNA fragments. By DNA sequence analysis In plasmids pAB7, pAB8 and pAB19, the linker-DNA was found to be attached to the 3' end of the CAT engineering structural gene in each of the six open reading frames. .

Plasmids pAB 7, pAB 8 and pAB 1.9 were each treated with restriction enzyme sets. Digested with S1 exonuclease and incubated with S1 exonuclease. Phenoh After chloroform extraction and ethanol precipitation, these blunt-ended plasmids were The offspring was digested with 'raql, and a DNA fragment of approximately 750 base pairs was immediately released. these The fragment contains a complete CAT fusion structural gene with one Bgl site in six open reading frames. but lacks the cA Ts-promoter.

These CAT engineered genes were transferred to the trp promoter of cilasmid pCT54. (Mtezo, etc., Zoro Seeding National Academy) ・Science, v, , -Engineering x-■-((Pro, Natl, Acad, S ci, USA) 80.3671 to ro 675.1983). This plasmid is transferred The advantage of having a transcriptional terminator sequence is that high levels of expression can be achieved over this sequence. cloned downstream of the flow and trp bromotors and restricted to two genes.

Plasmid pcT54 was digested with EcoRI, and the 5' sticky end was DNA Hr + ) The Flenow fragment of merase I was used to fill t7. This molecule is controlled by the enzyme c1a1. The CAT1 fusion vector gene cart isolated above was isolated and subsequently dephosphorylated. We obtained a molecule that accepts ridges. This molecule is added to 6 times the molar amount of each GAT T gene group. - Ligate with tricine, then transform large intestine 1HBi01 to Nicole resistance fusion vector plasmids pCT 201, pCT 202 and I ) CT 203 (Figure 3) was obtained. (In these six cases, pCT 54 EcoRI sites were reshaped).

Cilasmid vector pCT206 was cut with HindI and Ta. This yields plasmid DNA with Hlndl [sticky ends, and then It was smoothed with DNA polymerase.

Next, the generated plasmid DNA was further cut with Bgl■, and the Bgl■ sticky end was A DNA molecule with a Y-slip end was obtained.

The generated DNA was transformed into plasmid ph, T-853 Bgl, l-Pvu1 1 fragment (see Figures 1 and 6), a plasmid circular molecule was obtained. these pluses The mido molecule was transformed into E. coli HB101 cells, and E. coli HB IQ1/ The pCAT-cGRP transformant was grown in a medium containing 77 bisiliy<　10047ml) were grown and selected. By culturing, Escherichia coli HB IQ 1/pcAT- CGR cells were analyzed by SDS polyacrylamide single cell electrophoresis and Komatsushi blue staining. By color (Commassieblue stainlng) (Figure 4a), or pulse cells with 35S methionine for 1 min followed by SDS polyacrylamide. As judged by del electrophoresis followed by autoradiography (Figure 4b), the expected produced a fusion protein with a large size (Mtezo, Zoei, Nis, etc., PNA S (1983) 80.6671-3675).

In the pCAT-CGRP configuration, compared to the CAT protein alone, the new protein Figure 4 (a), 4 (11)), lanes 1, 2) - Figure 4 (a) ), 4(b), see Lane Ro. Lane M shows molecular weight marker proteins and The molecular weight of each is shown.

Evidence that the produced fusion protein contains the CORP methane sequence is HB101/ 1) CAT - Measured by radioimmunoassay of coRp cell extract.

Take 50ml of cultured cells and add lysozyme/sodium deoxycholate mixture ( 5ml) and treated with DNase for 30 minutes at 5°C (Mtage J. Nis et al., PNAS (1983), 80.3671-6675). Equal capacity 0.1M) Liss hydrochloric acid pH 8.0, 0.1mM EDT, A, 5% ( V/) Add glycol, h) Existence of CGRP sequence is excluded It was measured using the product. Radioimmunoassay was performed on skin 0'5 M phosphate buff-p) 17.4 in a final volume of 400 μl (Gargis Nis I et al., Journa Le, Endoc IJ/D G (J, Finclocrinol,) 78 , 372-382). Tyr-(CGRP'amino acid 25-'37) Using known techniques, antiserum was made in rabbits against 1251Tyr-7mir. (CGRP amino acids 25-57)-amide tracer, Conjugated to Min (Raichi Lin-M, 1980, Method Enzymology) (Meth, Enz7mo1.) 70, 159-165).

Tracers were iodinated using the Chloramine T method of Hunter and Greenwood.

It can be seen from Figure 5 that the HBl 01/pCAT-cGRP protein extract 1 by a displacement curve similar to that of the human QC) RP standard (chemically synthesized). :10000 serum dilution was used to replace the tracer (5C100cpm) . Predigested with trypsin (0.5 mp-/ml) overnight at 67 °C, followed by No displacement was observed using a protein extract in which trypsin was inactivated with silole. Ta. Rabbit 1gG was quantitatively precipitated using goat anti-rabbit serum, and carrier After adding 1 μl of pre-immunized rabbit serum as Leib R.K. et al. 1.1976゜Biochemistry Journal (Bi ochem, J.) 160.57-74), and precipitate 1251 was treated with γ-cure. It was quantified by counting. This is HB101/I)CAT-CGRPK (more) Demonstrate the production of the CGRP peptide sequence.

The composition of pOAT-CGRP cilasmid was checked by 5cal digestion.

This is due to the expected amount of DNA, which is larger than the corresponding band obtained from pCT203. A band was obtained on the gel. Plasmid composition can also be checked by Hael digestion. I clicked. p'h'r-8,5F3-derived Bgl]l-PvulIcDN The A'Ifr piece contains a Hae■ site that is not present in pOT203, and the revolver contains the expected A cDNA band of a certain size was shown.

The fusion protein produced by pOAT-CGRP has a carboxyl terminus and an amine. It consists of the amino acid sequence of CORP with additional amino acid residues at the end (Figure 1).

The amino terminus contains the sequence Lys-Arg immediately before QGRP. This is a clostri provides a pine cleavage site, but in view of the latent clostopain site within CGRP, must be taken into consideration. Other unique cleavage sites can be used.

Production of QGRP by chemical synthesis ) QGRP and PDA-4 are manufactured by Serotic Co., Ltd. 244-250 pass load, speed by Rowe, Parkshire, Nis.L.I., 407 United Kingdom, or Peninsula Lab. Latry, Inc., 61 Taylor Way, Belmont, California 94002 USA Synthesized by. Standard techniques for peptide synthesis can be used, e.g. Field solid-phase peptide synthesis or so-called FMOQ operation (“solid-phase peptide synthesis” 2-Re-evaluation (5oll Phase Peptlde 5ynthesis-8 Reassessment) J 7-/I/-Shi x Part-Monki Natural Endocrinology Edition, Matsukintyre and Zerke, Enzevier (19 77) 43-56: E.7”i-) N et al., C.C.N.C.M.C. J, C, S, C! hem, comm, ) (198j) 1151-11 52; and G. Paraney and R. B. Merrifield, The Peptide. (The Peptides), Yi Gross and J.J. -, Akademi Press, New York (1980)).

Cardiovascular effects of human CGRP Using the amino acid sequence predicted by nucleotide sequence analysis of phT-858, Then, amidated human CGRP was chemically synthesized and then subjected to ion/reversed phase chromatography. It was purified using a combination of Final h) (:! GRP preparations were determined by mass spectrometry. It was pure and a single ion was observed (Mr3786). The inventor has developed this preparation Synthetic pine of similar purity C! compared to GRP preparations.

Dissection of 4 to 6 Spray Good Urii Rats (285-31519) is performed using Bendopa. The animals were anesthetized with Rubiton (60 to 9-↓, intraperitoneally). Trachea, left carotid artery and left neck A cannula was inserted into the vein. Blood pressure is Stitham P23ID pressure drink The average arterial pressure was recorded from the carotid artery on a glass polygraph using a was induced. The heart rate was measured using a tachograph (glass model) guided by blood pressure signals. 7P4) Measured by IC. h) CGRP is crudely produced from horse chestnut fat and then purified. However, in the latter case, it was obtained in purified form from Peninsula Labora.

Purification Run) C0RP was of the same origin. All synthetic preparations are by mass (M-Scan) to confirm structure and purity before use. H) CGR P, rat CGRP, propatool hydrochloride (Sigma), mepyramine maleate (Sigma), cimetidine hydrochloride (Niss K & F), histamine syphosphate ( Sigma) was dissolved in 0.9 w/v saline. All compounds were mepyramine All drugs were administered intravenously, except for S.C. Saline solution (・X human C () RP (0) Yobi Rat C! GRP (△) was cumulatively applied to rats at 2-minute intervals in Q, 1ml: volume. It was administered in multiple doses.

The peak drop in mean arterial pressure (MAP) and the heart rate (HR) after 2 minutes were measured. . Administration of saline does not change MAP or HR, CGRP and rat cGRP Both induced a dose-dependent decrease in MAP and an increase in HR. h) [:! GRP 5 minutes ago, propranolol, 6.4 μmol of 9-1 (b) prevented the increase in mood frequency. I didn't get it. Metramine (12,4 μmol kg−1) and cimetidine (30 min infusion) 59.51r+le kg-1':1 (ili) is hypoglycemia for histyl6 Although pressure sensitivity was arbitrarily lowered (10-ε1 to 10-5 molar ky'), i did not significantly change the hypothermia effect. Even after injecting saline seven times during the experiment, the Significantly altered MAP or HR in the presence of lopranolol or mevilamine and cimetidine. I couldn't.

In rats anesthetized with bendoparbitone, intravenous Hi-C0RP significantly lowered blood pressure. It causes a rapid drug-related decline, reaching a maximum within 1 minute and reaching o,2snmol kg- A dose of 1 showed a 50% decrease in blood pressure. This is shown in Figure 5)CORP(0,2 The results were not significantly different from those obtained with p-mouth, 05, t-te strike). Hypotension showed a small but significant relationship with increased heart rate. The drop in extraction pressure is Basic peptides (which may be indirectly caused by C) lead to the release of histamine. (Gos A (197ro) Histamine and antihistamine istamine andantihistamines) (Shahif-・ M), Volume 1, Encyclopedia of Pharmacology and Treatment (Int, Encyclopedia a of Pharmacology and Thera, peutics, Section 74゜25~4゜Pergamon Press, Oxford) Shikatsuhi) (Since IGRP is a relatively basic peptide, the present inventor Before administering the histamine H ale sezocu antagonist mepyramine and histamine H The effect of pretreatment with the 2-receptor antagonist cimetidine was tested. Antagonist is h) Hypotension sensitivity to CGRP K (Figure 6) or related tachycardia (data) (・) There was no significant effect on Ic. Increased sympathetic drive It was also suggested that CGRP may increase the number of mood swings (Fisher et al. ・El Cony, Kirakawa D-O, Riviere G.I., Ama La Nis G, Evans R.M., Rosenfeld M.G., P.E. Ill. W. D. & Brown M. R. (1983), Nature - (IJature), 305.534-536). The inventor has determined that the α-address noceptor antagonist propranolol (isoprenaline on the right side depending on the 210g unit) Human CGRP We investigated the possibility of jin by intravenous administration of . Basic mood than propranolol Although the number decreased significantly, cORP continued to cause tachycardia, and saline control treatment Compared to (Fig. 6), the limiting dose for this effect remained unchanged. These consequences As a result, the decrease in blood pressure and increase in heart rate caused by human c()RP are due to histemia. It is not mediated indirectly by the release of amines or cetacolamine. h) (: per 1 GRP) The sensitive period was measured by single drug administration. Human C0RP 1 nmol kg- ] changed the arterial pressure from 125 ± 7.6 mm Hg to 68.6 ± 9.3 mm Hg. The time required to lower blood pressure and restore blood pressure to 50% is 3-8 + 0.5 minutes. Ta.

Rat K anesthetized with the above phenobarbitone: Intravenous POA-40 Tests of cardiac transection showed a small but significant effect. As can be seen from Figure 7, 7 PDA-4 had 50-100 times lower potency than human C()RP.

Once, 1μI/kilohi1. CGRP has a steep mean arterial pressure of 50iT This resulted in a decrease in nHg and an increase in heart rate by 25-30 bpm. On the other hand, 10μg/ Kilo PDA-4 reduces mean arterial pressure by 15-20 mmHg and decreases heart rate by 1 This caused an increase of 5 to 20 bpm. The mechanism of action of PDA-4 was not tested. It was.

To eliminate the effects of reflexes or other factors that cause a drop in blood pressure in the body , h) The cardiac effects of C0RP were also studied in vitro.

Dunkin' no-tray guinea pig M (280-400,!7) twists the neck. The animal was killed and the blood was removed. Remove the heart 2, cut the right atrium into small pieces, and add Krebs solution (mM) NaCf 113, Kci4.7, CaCj! 22.5, KH2P O41-18.

NaHco325, MgSO4,i, 13% and glucose 11.1 It was set at 34°C and a tension of 0,5,9, and bubbled with 95:502 CO2. Ga Measuring atrial force and atrial velocity for equal purposes using a Las FT Q-filtration transducer and recorded on a glass polygraph. Human (・) or rat (Mu) Raise C0RP A single dose was administered randomly. In preliminary experiments, two consecutive doses of CGRPJ The response curve shows reproducible results L-0 Propranolol (○) (30 [ ] nM) or cimetidine (1 00zm) were each 9 in the same experiment. Neutralizes the effects of imprenaline (3-30nM) or histamine (500nM) It was peaceful. These antagonists suppress the increased atrial velocity and cardiac It did not significantly reduce tuft force. Antagonist alone reduced the basal rate of isolated atrium 5 (187 soil 9 b, min-1) or power (264 f - 34 mp).

In guinea pig right atrial preparations, human C0RP showed a concentration-dependent rate and force during contraction. showed an increase in survival (Fig. 8).

Consistent with the results obtained in vivo, propranolol or histamine H2-receptor - The concentration of the antagonist cimetidine that effectively blocks β-adrenoceptors is H) C0RP I didn't change my reaction to it. Of interest, rat C0RP is associated with atrial force (a trj, al force) increases - t h) has the same ability as CGRP but was approximately 10 times more capable at increasing atrial velocity (Figure 8). La) Increases in flooding/cluster velocity or force caused by C0RP can be blocked by propranolol. was not checked. Therefore, in rats and humans, CGRP is produced directly in isolated atria. The action is dependent on catecholamine and histamine receptor mechanisms. do not.

What the inventor's research has demonstrated is that human and rat CCI in anesthetized rats Peripheral cardiovascular effects of RP were observed for rat C0RP administered peripherally to conscious animals. Reports 1 to 1 are similar in that C0RP was used as an anesthetic in the inventor's experiment. Due to the probable dullness of cardiovascular reflexes, the concentration may be significantly increased in intentional preparations. Increased (Morrison, G.L., Walker, R.C., & Richer Dosun A.P. (1950) Arch Inter Pharmacodyne (Arc h, engineering nt.

Studies have shown that the vasodilatory effect of CGRP is mediated by histamine or catecholamines. It has been proven that there is no interference. Therefore, CGRP is a new scepter machine. Acts directly on the cardiovascular system due to its structure, and does not act through the release of other mediators. proves that. Similarly, it is not affected by propranolol or cimetidine. The activity of 0GRP on isolated atrium was determined by C0RP reactivity in cardiac tissue as well as in the vasculature. It supports the concept of a septa mechanism.

(in humans and rats) that lowered blood pressure in vivo and increased atrial contractility in vitro. Similar abilities of C0RP contrast with their various abilities when increasing the rate of atrial contraction. Comprehensive. Results obtained in vitro show that CGRP is probably a different Due to the presence of species or anesthetics, properties that were not clear in vivo, desirable chronotropic effects. This suggests that it has an effect (compared to inotropy). The inventor is a PDA-4 has been shown to have a hypotensive effect with simultaneous tachycardia when administered intravenously.

Diagnostic use In Figure 2, using the amino acid sequence predicted by CGRP, amidated CGR P and tyrosinated analogs; Tyr-(CGRP-) conjugated to ovalbumin Antibodies were raised in rabbits against amino acids 25-37)-amide (reitilin (1980), Menrod Enzym (Method, Enzym, ) 70. i'59-165).

1251 Tyr-(cGRP-amino acids 25-37)-binds amide Both demonstrated antigenicity as measured by potency. As mentioned above, the present inventor has provided the following information: : 180 cpm 125I in 400 μl test with serum dilution of 12,500 -Tyr-(cepp-'amino acids 25-37)-7 using midotracer We conducted a radioimmunoassay sensitive to the Inter (Engineering) C0RP/tube. For Tyr-(C0RP-amino acids 25-37)-amide, Antibodies were used. Using this test (Figure 9), this antibody Rosinated PDA-4, Katacalcin, Tyr-CGRP-(amino acid 1- 8) or salmon calcytosine, but the antibody does not cross-react with rat) C0RP. When titrated with increasing amounts of CGRP, the expected position The present inventors have proven that it shows a conversion curve. Using this test, human medullary thyroid Extract from adenocarcinoma tissue (Bennett H.P. et al., 1978, Pyochemist Lee Journal (Biochem, J,) 175, 1139-114 1) and tissue culture derived from human small cell carcinoma cell line (DMS 153) autopsy livers known to produce nutrient media and low levels of calcitonin. Cell lines derived from metastases (early lung) (Petotenzil et al. (1980)) - (Cancer) 45, 906-918), the present inventor has The presence of C0RP was identified. Tissue culture medium alone did not react with antiserum, and DMS5 Medium extracted from 3 cells is known to produce high levels of calcitonin. small cell carcinoma cell line (Sorenson et al., 1981, Cancer ) 47.1289-1296) had only a slight H) C0RP.

Parallel displacement curves were observed for MCT extract and DMS 153 media. normal Human plasma did not contain detectable amounts of C0RP within the scope of the study, and some MCT patients plasma also contains measurable amounts.

The present inventor has developed a peroxidase-antiperoxidase method (Sternberger L. ・A, 1979, Immunocytochemistry ) 2nd edition; Immunocytization at a light level in paraffin wax embedded with medullary thyroid cancer tissue Human CC) RP-producing cells were generated using scientific methods, and these antibodies were classified according to the classification of human tumor pathology. It has proven its diagnostic application in the body. All ports! J (A)-containing RNA was isolated from two different Isolated from medullary thyroid carcinoma and DMS 53 and 153 cell lines (Allison et al. (1981) Biochem'Journal (Biochem, J.) 199, 725 -731 and Hall et al., 1979, Nature 3 The distribution of CORP and intron specificity transfer (see Figure 1) was 1.1% (w /v) Electrophoresis in agarose del, followed by Biodyn e) Study by RNA blotting including transfer to membrane and separation (Tiller G.P. et al., 1984, Biochem Journal: r, ) 219, 223-231) o In another experiment, Bgl II / Pst I 32 p-label calcitonin specific cDNA fragment (Sp, A c, 3.2 X I Q8cpm / aj9'), Bgl l / Ps tl CGRP 1% isomeric cDNA fragment (Sp, Ac, 7.9K 10” cpm/4) and BgI II/Bgl II “Intro 7” Specificity cDNA fragment (Sp, Ac, 2-8 X IQ” cpm 78g ) was used to probe this membrane, see Figure 1. After washing the filter, auto I went to radiography. This result is consistent with the RIA data and shows that calcitonin and C GRP mRNA species are expressed at various levels in cell lines and tumors and are associated with calcitonin. and CGRP mRNA are probably synthesized from normal transcripts by various procera song pathways. It was verified that the cells were processed (Fig. 10). In addition, the intron specificity sequence is No mature process calcitonin or CGRP mRNA species were present.

Normal placental DNA and the patient's MTC tissue DNA and blood lymphocyte DNA were used as restriction extracts. After digestion with endonuclease (Figure 11), the denominator of the human CGRP gene sequence Research on objects also produces differences in genetic composition (gθne, orga, n1sation). I did. Therefore, each DNA sample 204 was restricted with BamHI and the fragments were isolated at 1% (v y/v) Separate the cells according to size by electrolysis using agarose gel, and perform genetic screening. plotted on the lath membrane (NEN), and then plotted on the specific activity of IQ”cpm/μg. using the labeled Bgl II/Pst l CGRP-specific cDNA probe. I probed. Hybridization is 50mm) Squirrel-HcJ, pt (7,5 medium 50% (v/v) formamide, 1 t (w/v) SDS % 10% (w/v) Dextran sulfate was carried out overnight. Filters are sequentially 2x SSC/RTP, 2ys’sc, 1% (w/v) SDS, 65°C7 30 minutes, and washed in 0.1X SBC, 65°C/15 minutes, then 48 hours. Submitted to autoradiograph. This is true for all DNA samples... Prove the single major band (2,8,Kb) and minor band (2,6Kb) of (Fig. 11), but there was an additional minor band (3.0 Kb) in placental DNA. . Therefore, using the CGRP-specific cDNA probe, we I checked the sequence. In this case, the gene is homologous to the probe, as opposed to being homologous. show. These findings may be useful in studying gene rearrangements in tumor tissue. This demonstrates the diagnostic value of the probe. In this case, a family of medullary thyroid cancers Using the KcoFuF gene probe to study binding restriction enzyme polymorphisms of the form Special publication date 'Q GO-501562 (11) The present invention and the possibility of Research on the cardiovascular activity of cGRP uses the rat calcitonin gene as a model system. (Amara Nis G, Jonah) S. V., Rosenfeld M. G., Onzo E. Nis & Evans. R.M. (1982), Nature, 298°241 ]-244) (Rosenfeld M.G., Marmotsud G.J., Amara Nis G, Swanson El Tahryu, Souchenko P.I. -, Rivière J, Vert W. Duffry & Evans R. ・M (1983) Nativier G.E., Amara N.G., Eva. Nth R.M., Rosenfeld M.C., Baer/”-1 Rieux ・Doubliny table Brown M.R. (1983) Nature (Natur s), 3o5゜554-'536). The inventors have investigated human thyroid and lung cancer. The CGRP mRNA sequence and calcitonin were identified.

The findings are based on human C0RP or its fragments, lung cancer cell lines, and medullary thyroid cancer cells. It was identified using antibodies against cGRP in tissue and plasma. Therefore, plasma C0R Measuring P levels, historical tissues using in-situ hybrid or immunocytochemical techniques testing, or testing of gene structure and expression using DNA or RNA blotting. It is useful in the management of medullary thyroid cancer (Hill C. Nis, Ipanets M. L., Samaan F.A., Ahaan M.S.E. & Clark R.E. (1973), and has also been associated with lung cancer and abnormal calcitonin gene expression. It is also useful in the management of known diseases such as osteoporosis.

Amidated H) C0RP peptide PDA-4 has an effect on the cardiovascular system, causing cardiac contraction. The knowledge of the present invention that it causes an increase in speed and force and has a blood pressure lowering effect is due to the fact that The results suggest a role for this peptide in the clinical management of pressure I-.

The peripheral effects of cGRP are due to catecholamine β-lysezota and histamine receptors. The present invention proves that it is independent of the data source. Sudden response to hypotension caused by peptides Considering the rapid onset, C0RP is a new υ receptor that does not depend on other mediators. - The effect of the present invention is consistent with the view that it can directly act on the cardiovascular system through a mechanism .

Car L Shi U:, > ]-I Aja 1” 1□ FIG. 1 Engraving (81 of them unchanged) FI6.2 Symbol Sho GO-5015G2θ? ) ・Government・Tachisue FIG.8 6 rt-y)-:y-, (-q91), x-g-”i>t-p 嘗a, 'l'7oo -'r□□ F; IY">R-btl to t) 1 hair! Shi JA ,,ノI71・N,-Toki FIG. 10 i ii iii Procedural amendments issued by Kui) 2nd day of the month of 1985.

Commissioner of the Patent Office 1.Display of the incident 2. Invention 0 name i'#-)', 5e 11. -1, two, host organism, its Manufacturing method and diagnostic agent 3. Person who makes corrections Relationship with tenant farming Patent applicant Showa year, month, day Procedural amendment (formality) Date: June 1985 Commissioner of the Patent Office 3. Person who makes corrections Relationship to the incident: Patent applicant 5. Date of amendment order Showa BO year month/day 6. Number of inventions increased by amendment 7. Subject of correction hD of translation of drawings (no change in content) international search report InLun5+lo, al AppHca+lon N. PCT/GB 841 Continuation of 00210 page 1 QInt, CI, 4 Identification symbol Office serial number priority claim 019 November 1 Japanese [phase] United Kingdom (GB) [stock] 8329093 @ originator Nidoprusk, Mark Robbie - United Kingdom Dub 0 Applicant Nidprutsk, Mark Robbie Dove Biochemist, Greece Special edition Showa 60-501562 (16) 7゛Riyu 1P 7PN, London, Mortimer (no street address) Middlesey X Hospital Medel, Sa Courtauld Institute Obry Inside 7゛Riyu 1P 7PN, London, Mortimer (no street address) Middlesey X Hospital Medel, Sa Courtauld Institute of Li inside

Claims (1)

[Claims] 1. Human lucitotoninogene-related peptide. 2 Structure Ala-Cy 5-As p-Thr-Ala-Thr-Cy s-Va 1- Thr-Hi s-Arg--Leu-Ala-Gly-Leu-Le u-8er-Arg-8er-Gly-Gly--Val, -Va 1-Ly s-Asn-As n-Ph e-Val, -Pro-Thr-Asn--Va l, -G], y-3er-Lys-Ala-Phe-amide (1). zute) pen 0 chido. 71F), the peptide according to the second addition, if used as station 2, if't'l longer range. 4 High aspect 1=therapeutic treatment (Claim 1) (1 description θ) peptide. 5.Claim 1.]) Benochide ojyohi [2 sho. j] add a functional excipient; 1.・7.Composition. 6 At least the following amino acid sequence 1] AJa, -Cys-Asp- Thr-Ala-Thr-Cys-Val-Thr-'rjic--Arg-L eu-Ala -Gly -Leu-T, +0L1-8 C1r -Ar g-Ser　-t)ly--01y-Val　-Val-LyS-Asn-A sn-Phe-'Val-Pro--Thr-AG n-va 1-G 1y -8er-LyS-A 1a, -Phe-R1 (11) (in the formula, r' is -H Or amino acid donation residue. :, two conversion n] resistant peptides. 8 681. The Ray peptide of claim 681, wherein ZR' is -Gly. 8. Production of peptide having structure (1) d. ◇ 1. Claim 6 or a peptide described in paragraph 7 ()), which contains a gene encoding an intermediate peptide. Vector shape 'l' 4+! , the transformed host organism is cultured to produce intermediate peptides. 1:), then converting R' to amide (right -9 and 14'a), -1 notation Law. 9. Intermediate peptides are produced at high levels in 41 transformed hosts. At least -F + (lv) kun white tr1:t, and claim 68' ! Also (No. 71 above) j; ] 1. −, the fusion protein association, arcminute, l, −(=, 5i′1 sought σ゛ range 6th term Or the 7th! Li g [: il & (4 + bezochidoi +” 7:j l), - Fortune-telling θ) plane 2 ptl gf4. Rlrj urgent request: ji)9 / idiot θ -10 transformed host 1 object L#, -t: Bar 14: L 5 I ', at least some proteins, t°C, 9Lυ', 6th column ( 1.1.1. The second protein Ifj, Tobezobudo is kasho Or, by enzymatically selectively decomposing the vine bond, the fusion tongue can be isolated. 11 The bond is -Met-1-()]Il-OL-by3-Ar(,y-glass If selected, the crystallization range is 'Ofil!' iL: 1st evening/cl fi, ) 12 structure j11, (1) is present, peptide (7) Dli J/, :,-,,,,l:,r,,-o,e(,at least the lower 8 [-1 amino alcohol array Arg-E-A1a-G], n-Lys7Arg-Ala-Qys -Asp--Thr-Ala-Thr-Cys-Val-Thr-His-8r g-Leu-Ala--G 1y-L eu-Leu-8er-Arg-5er -Gly-Gly-Val-Val-Lye--Asn-Asn-Phe-Va Intermediate peptide containing l-Pro-Thr-Asn-Val-Gly-8er By culturing a host eukaryotic organism transformed with a vector containing a gene encoding , obtaining an intermediate peptide and processing this intermediate peptide by a host organism; The above method characterized in that the peptide is then isolated. 16 The following amino acid sequence. Tetrapeptide with Asp-Leu-Gln-8 la (lv). 14. The tetrapeptide according to claim 16, which is used as a medicine. 15. The tetrapeptide according to claim 13, which is used for the treatment of hypertension. 16. Tetrapeptide according to claim 13 and pharmaceutically acceptable excipients A pharmaceutical composition comprising an agent. 17 Any one of claims 1, 2, 6, 10 to 16 A gene encoding the peptide described in . 181) Amino acids 1-67. 11) Amino acids -6 to ---7, 111) Amino acids +6 to +9 or iv) Amino acids -7 to +9 The gene described in the lower part of FIG. 2 encoding. 19. A vector comprising the gene listed in claim 17 or 18. 2. A host organism transformed with the vector according to claim 19. 21 A peptide of structure I or a portion thereof containing an antigenicity determinant; or portions thereof have a detectable label attached thereto. 22 A peptide of structure (1) or a portion thereof having an antigenicity determinant, and a peptide or a portion thereof. contains an arbitrarily labeled tyrosine amino acid residue at 125. peptide. Structure, with Tyr arbitrarily labeled at 23.1251. Tyr-Asn-Asn-Phe-Val-Pro-Thr-Asn--Val -Gly-8er-Lys-Ala-Phe-amide, claim 2 Peptide according to item 2. 24, 1251 with Tyr arbitrarily labeled, structure: Tyr-Ala-Cys- Asp-Th, r-Al-a-Thr-Cys-Val, Claim No. The peptide according to item 22. Tyrosinated PDA arbitrarily labeled with tyrosine in 25, 1'25 process -4゜ 26 An antibody having specificity for the antigenic determinant of the peptide of structure I. 2Z An antibody having specificity for the antigenic determinant of the peptide having the sequence ■. 28 15 or more nucleic acids selected from the nucleotide sequences 1 to 1256 shown in Figure 2 DNA hybrid probe with leotide sequence. 29 A claimant having a sequence selected from the nucleotide sequences 1 to 725 shown in Figure 2. The DNA hybrid probe according to item 28. 30 It has a sequence selected from the 726-1256 nucleotide sequence shown in Figure 2. The DNA hybrid probe according to claim 28. Engraving (no changes to the content)