WO2002002594A1 - Peptide pour reactifs utilises dans des tests de gestation porcine - Google Patents

Peptide pour reactifs utilises dans des tests de gestation porcine Download PDF

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Publication number
WO2002002594A1
WO2002002594A1 PCT/JP2001/005867 JP0105867W WO0202594A1 WO 2002002594 A1 WO2002002594 A1 WO 2002002594A1 JP 0105867 W JP0105867 W JP 0105867W WO 0202594 A1 WO0202594 A1 WO 0202594A1
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WO
WIPO (PCT)
Prior art keywords
peptide
swine
pregnancy test
pig
pregnancy
Prior art date
Application number
PCT/JP2001/005867
Other languages
English (en)
Japanese (ja)
Inventor
Masaru Sakaban
Kazunari Yamano
Koichi Doen
Original Assignee
Zac Corp.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zac Corp. filed Critical Zac Corp.
Publication of WO2002002594A1 publication Critical patent/WO2002002594A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones

Definitions

  • the present invention relates to a pig pregnancy test reagent peptide and a pig pregnancy test reagent used for pregnancy test of a sow when breeding is managed in the pig raising industry.
  • Conventional technology relates to a pig pregnancy test reagent peptide and a pig pregnancy test reagent used for pregnancy test of a sow when breeding is managed in the pig raising industry.
  • Pigs (Su s scr afa dome sticus s and other closely related species, hereinafter referred to as pigs) are widely used for food or medical and research purposes because of their strong fertility and fast growing livestock. Has been offered to.
  • Pigs can be used for breeding at any time of the year, with males and females 10 months after birth and weighing about 11.5 kg, but in Japan they are expected to give birth in March or September when the climate is good. It is preferable to breed by artificial insemination around the month or November.
  • the time for mating is as short as about half a day to one day after entering the estrus period of the 21-day cycle, so it is important to ensure that the fertilization is not missed and that the fertilization is confirmed.
  • the conventional method of confirming conception is to observe the natural breeding behavior of boars, but there is also a more scientific method that uses ultrasonic diagnostic equipment.
  • estrone sulfate was not found in the blood of non-pregnant pigs, but was found in the blood of pregnant pigs.
  • estrone it was common to first detect sulfate-bound estrone by liquid chromatography.
  • the equipment cost is high because the diagnostic apparatus for humans is diverted, and the diagnostic method using such an apparatus is also required.
  • the work is complicated, and it is difficult to make accurate judgments without a skilled person.
  • an object of the present invention is to solve the above-mentioned problems and to provide a peptide (required component) for a reagent capable of performing a pig pregnancy test without error in diagnosis by a relatively simple method.
  • An object of the present invention is to provide a pig pregnancy test reagent which can easily and reliably diagnose pregnancy or non-pregnancy using the method. Means for solving the problem
  • the present invention provides a pregnancy test for a pig comprising a peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and having a binding property to estrone produced by a pregnant pig. It was a peptide for a reagent.
  • the peptides of the present invention configured as described above were estimated by using the special computer software by the inventors of the present application, and were prepared using six specific amino acids synthesized using the amino acid synthesis method. It consists of a short peptide of the sequence, which has the binding property to ester D produced by pregnant pigs. It is a short peptide to which a labeling substance can be attached. Therefore, it is possible to easily find estrone produced by pregnant pigs by utilizing such a binding reaction between the peptide and estrone (eg, agglutination reaction).
  • the peptide of the present invention is composed of short peptides each having a specific amino acid sequence as a unit, it can be efficiently produced by a well-known chemical synthesis method or the like. It also has the property.
  • the invention according to the pig pregnancy test reagent of the present application comprises the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and has a binding property to estrone produced by a pregnant pig,
  • the invention of the peptide for a pig pregnancy test reagent of the present invention provides a method for preparing a lysine (Lys) or two or more lysines (Lys) linked to an end of a branched lysine. And a pig for a pig pregnancy test reagent obtained by concatenating the peptide of the amino acid sequence represented by SEQ ID NO: 1 or a pig containing the above-mentioned pig pregnancy test reagent peptide as an essential component.
  • peptide of the present invention configured as described c above is to that a pregnancy test reagent, binding the pregnancy swine production Est opening on to the N-terminus of the branched peptide in which one or more lysine (Lys) are linked
  • a pregnancy diagnostic reagent for pigs containing such peptides as an essential component Is relatively simple It can simply be used to diagnose pregnancy or non-pregnancy, making it a diagnostic reagent with few errors.
  • FIG. 1 is a chart showing the amino acid sequence of a branched, swine-produced ester-binding peptide.
  • a peptide comprising the amino acid sequence (Ala Leu Asp Tyr Tyr Thr) represented by SEQ ID NO: 1 in the sequence listing can be produced by a well-known peptide production method such as a chemical synthesis method. Specifically, it can be produced by techniques such as chemical synthesis and genetic modification.
  • the C-terminal amino acid of the peptide to be synthesized is immobilized on an insoluble support such as cross-linked polystyrene, and this is used as a starting point.
  • N-—t-butoxycarpolinylated amino acid (Boc-amino acid) or N- ⁇ -9-fluorenylmethoxycarbonylated amino acid (Fmoc-amino acid) using a commercially available synthesizer controlled by a microcomputer
  • the protecting peptide is appropriately removed to extend the required peptide chain in order.
  • the manufacturers or sales companies of peptide synthesizers include Pharmacia Biotech, Perkin-Elma Japan, Aroka, and Shimadzu.
  • a peptide is synthesized using Boc-Lys (Boc) or Fmoc-Lys (Fmoc) in an insoluble carrier such as cross-linked polystyrene.
  • Boc is a tertiary butyloxycarbonyl group
  • Fmoc is a fluorenylmethoxycarbonyl group.
  • the Boc method or the Fmoc method which is a method of synthesizing the peptide by a usual solid phase synthesis method.
  • the amino group must be acetylated to prevent the subsequent elongation of the peptide chain and to prevent the formation of peptides lacking amino acids in the middle.
  • the branched peptide shown in FIG. 1 is obtained by linking the aforementioned predetermined amino acid sequences via 15 lysines (Lys).
  • lysines Lys
  • SEQ ID NO: 1 the case where two sets of peptides represented by SEQ ID NO: 1 were bound to one lysine is shown in SEQ ID NO: 2 in the sequence listing.
  • a core in which lysines are linked in a linear or branched manner is used.
  • n l, 3, 7, and 15, 2, 4, 8, and 16 peptides can be connected in a branched manner.
  • the number of lysines used for the core of the peptide linked to two or more may be two or more, but it is preferable to link about 2 to 15 lysines. Even if a peptide with 16 or more lysines is prepared, it reacts more sensitively This is because the reagent may not be obtained, and the production cost may be high, and the practicality may be lost.
  • the labeling substance attached to the N-terminus of the peptide as described above should be able to easily identify whether or not pregnant pigs have the ostium produced in blood, saliva or other body fluids.
  • a well-known peptide-binding label can be used so that the conjugate can be observed with the naked eye or a colorimeter by aggregation, precipitation, or luminescence (fluorescence).
  • a fluorescent labeling substance can be cross-linked to the N-terminus of SEQ ID NO: 1 or 2, and the binding substance to pig estrone in the sample can be directly observed with a fluorescent tube. Colors such as green, yellow, and red can be selected as the fluorescent labeling substance.
  • Commercially available auramin (Muto Chemical Co., Ltd.), erythrin, fluorescin isothiocyanate (FITC) dye, etc. can be used. it can.
  • Pyotin was cross-linked to the N-terminus of SEQ ID NO: 1 or 2, and
  • the pregnancy test reagent for swine can be used to diagnose pregnancy relatively easily by recognizing an appropriate label that has been set in advance, and is a diagnostic reagent for swine pregnancy with few errors in the diagnosis.
  • a functional group was introduced into a polystyrene resin crosslinked with divinylbenzene.
  • resin hydroxymethylphenylacetamide resin (also called PAM resin) or 4-methylbenzhydrylamine resin
  • PAM resin hydroxymethylphenylacetamide resin
  • 4-methylbenzhydrylamine resin Use an automatic peptide synthesizer that uses Boc-Lys (Boc). Using.
  • Boc—A1a—PAM resin 0.03 mmo1 (1/16 of 0.5 mmol) and Boc—Lys (Boc) -OH0.09 mmol was coupled with the OP.
  • Boc-Lys (Boc) -0 HO.09mmol Boc-Lys (Boc) -OH0.18mmo1 was coupled.
  • oc—Lys (Boc) —OH 0.36 mmol and 0.72 mmo1 were sequentially coupled.
  • a branched peptide core in which 15 lysines (Lys) were linked was synthesized.
  • arginine (Arg) was immobilized by the automatic peptide synthesizer, and a short peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing was added to the 16 N-terminals of the core by the well-known Boc method. Then, a 16-fold branched peptide was prepared by binding 16 units of this peptide, and the protecting group was removed and the peptide was eliminated with a solvent.
  • predetermined amino acids are linked to the resin from the C-terminus by repeating the following mechanical operations (1) to (4). The volume of the solution during the operation is about 5 to 10 m1.
  • the protecting group of the amino acid side chain was deprotected with hydrogen fluoride (HF) simultaneously with the cleavage from the resin. That is, 500 mg of resin, 1 ml of m-cresol, 0.7 ml of ethanediol, and 0.5 ml of dimethyl sulfide were placed in an HF reaction tube. Attached to the HF device. The reaction tube was cooled to ⁇ 78 under reduced pressure, and HF 10 ml was introduced into the reaction tube. After stirring at 0 ° C for 1 hour, HF was distilled off. Anhydrous ether was added and the residue was transferred to a glass filter and washed well with ether dichloromethane. The peptide was dissolved in 10% aqueous acetic acid, and the filtrate was freeze-dried.
  • HF hydrogen fluoride
  • the obtained peptide was purified by high performance liquid chromatography (HPLC) using a reversed phase column.
  • HP LC high performance liquid chromatography
  • the conditions for HP LC were as follows: Wako Pure Chemical Industries, a column with an inner diameter of ⁇ 4.6 mm and a length of 25 Omm (Wakosi Le 2, 5C18 AR) packed with C18 silica gel with a particle size of 5 ⁇ m.
  • the peptide was eluted using a 0.1% aqueous solution of tetrafluoroacetic acid (TFA) containing 5% acetonitrile (CH3CN) as the initial solvent.
  • TFA tetrafluoroacetic acid
  • CH3CN 5% acetonitrile
  • Elution was performed for 30 minutes at a flow rate of 1.5 ml / min by temporarily setting a concentration gradient in the range.
  • the venous blood of each of the sows or one of the comparison sows which were distinguished from pregnant pigs and non-pregnant pigs by ultrasonic diagnostic method, was aseptically collected with a syringe at a volume of 10 m1.
  • the lath test tube was transferred from the 15 ml syringe for centrifugation to the centrifuge, and centrifuged at a speed of 300 rpm for 15 minutes, and the obtained serum was collected in another test tube.
  • the obtained serum of each pig was diluted with a phosphate buffer (pH 7.4) (manufactured by Nippon Suisan Kaisha, Ltd.) to dilute it 10-fold and 100-fold.
  • the peptide for the pig pregnancy test reagent of the example in which the peptide of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was linked was 10-fold or 100-fold phosphate-linked.
  • the pig pregnancy test reagent diluted with the buffer solution was agglutinated at any dilution ratio, and it was a pregnancy diagnosis for pigs that could be reliably diagnosed and had no measurement error.
  • a peptide derivative FITC-ALDYYT-K (molecular weight: 744.342) was produced by binding a fluorescent labeling substance, fluorescin-isothiothionate (FITC), to the N-terminus of the obtained peptide, which was used as follows.
  • FITC fluorescin-isothiothionate
  • the invention relating to an ester-binding peptide produced by a pregnant pig comprises a peptide having a specific amino acid sequence, and has an ester bound to the C-terminus of the peptide and a labeling substance bound to the N-terminus. Therefore, there is an advantage that this method makes it possible to easily find a pregnant pig-produced ostium not only from pig blood but also from body fluids such as saliva.
  • an essential component is a peptide derivative which binds an enzyme or a coloring substance to the N-terminal of the peptide having a specific amino acid sequence and has a binding property with a pregnant swine produced by swine.
  • the pregnant pig-producing ester has the advantage that it is possible to easily detect the pregnancy. There is an advantage that it can be done.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Endocrinology (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un peptide permettant de tester avec précision la gestation porcine selon une méthode relativement simple, ainsi que les réactifs de ce test permettant un diagnostic facile et fiable. L'invention concerne en particulier un peptide comprenant la séquence aminoacide représentée par SEQ ID NO : 1 dans l'ordre des séquences et étant capable de se lier à l'oestrone produite par une truie gravide et, de ce fait, utilisable comme peptide pour des réactifs pour tests de gestation porcine. L'invention concerne également des réactifs pour tests de gestation porcine, contenant, comme ingrédient actif, un dérivé peptidique édifié en liant un marqueur à la terminaison N du peptide précité. Du fait qu'un peptide à chaîne courte ayant une séquence aminoacide spécifique est capable de se lier à l'oestrone produite par une truie gravide, l'oestrone peut se lier à la terminaison C de ce peptide pour des réactifs de test de gestation porcine, et un marqueur peut être lié à la terminaison N du peptide. En utilisant un tel réactif pour le test, on peut facilement juger, en examinant le marqueur, si le réactif contient ou non de l'oestrone produite par une truie gravide. L'invention permet ainsi d'obtenir des réactifs pour des tests de gestation porcine au cours desquels la gestation peut être diagnostiquée relativement facilement avec une faible marge d'erreur.
PCT/JP2001/005867 2000-07-05 2001-07-05 Peptide pour reactifs utilises dans des tests de gestation porcine WO2002002594A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000203995A JP3738175B2 (ja) 2000-07-05 2000-07-05 豚の妊娠検査試薬用ペプチドおよび豚の妊娠検査試薬
JP2000-203995 2000-07-05

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01109262A (ja) * 1987-10-21 1989-04-26 Eiken Kagaku Kk 免疫測定用抗体及びそれを用いる免疫測定法
EP0328403A2 (fr) * 1988-02-12 1989-08-16 United Biomedical Inc. Peptides synthétiques relatifs à la protéine HIV-GP120-env. et leur application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01109262A (ja) * 1987-10-21 1989-04-26 Eiken Kagaku Kk 免疫測定用抗体及びそれを用いる免疫測定法
EP0328403A2 (fr) * 1988-02-12 1989-08-16 United Biomedical Inc. Peptides synthétiques relatifs à la protéine HIV-GP120-env. et leur application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DAVID J. YANG ET AL.: "Diagnostic and therapeutic potential of poly(benzyl L-glutamate)", J. PHARM. SCI., vol. 83, no. 3, 1994, pages 328 - 331, XP002947229 *

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JP2002187898A (ja) 2002-07-05
JP3738175B2 (ja) 2006-01-25

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