WO2002002594A1

WO2002002594A1 - Peptide for swine pregnancy test reagents and swine pregnancy test reagents

Info

Publication number: WO2002002594A1
Application number: PCT/JP2001/005867
Authority: WO
Inventors: Masaru Sakaban; Kazunari Yamano; Koichi Doen
Original assignee: Zac Corp.
Priority date: 2000-07-05
Filing date: 2001-07-05
Publication date: 2002-01-10
Also published as: JP2002187898A; JP3738175B2

Abstract

A peptide whereby swine pregnancy can be accurately tested by a relatively simple method and swine pregnancy test reagents enabling convenient and sure diagnosis. A peptide comprising the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing and being capable of binding to estrone produced by a pregnant pig which is usable as a peptide for swine pregnancy test reagents; and swine pregnancy test reagents containing as the active ingredient a peptide derivative constructed by binding a marker to the N-terminus of the above peptide. Since a short peptide having a specific amino acid sequence is capable of binding to estrone produced by a pregnant pig, estrone can be bonded to the C-terminus of this peptide for swine pregnancy test reagents and a maker can be bonded to the N-terminus of the peptide. By using such a reagent for swine pregnancy test, it can be easily judged whether or not the reagent contain estrone produced by a pregnant pig by examining the marker. Thus, it is possible to provide swine pregnancy test reagents by which pregnancy can be diagnosed relatively easily with little errors.

Description

Description Veptides for pig pregnancy test reagents and pig pregnancy test reagents Technical field to which the invention pertains

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

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.

In addition, according to Robertson's announcement on pig estrus hormones in 1978, estrone sulfate was not found in the blood of non-pregnant pigs, but was found in the blood of pregnant pigs. However, to detect estrone, it was common to first detect sulfate-bound estrone by liquid chromatography. Problems to be solved by the invention

However, in the above-described diagnostic method using the conventional ultrasonic diagnostic apparatus, 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.

'Ivy o

In addition, the above-mentioned conventional methods for detecting estrone require a long time in the extraction step and the fractionation step, and the cost of measuring equipment and other equipment tends to be high.In addition, if the accuracy of diagnosis is sufficiently high, I can't say.

Therefore, 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

In order to solve the above problems, 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).

In addition, since 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.

Further, in order to solve the above-mentioned problems, 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, This was a pig pregnancy test reagent containing as an active ingredient a pepti Byone derivative having a label attached to its N-terminus. Since this peptide derivative has a binding property to estrone produced by pregnant pigs and has a labeling substance such as an enzyme or a chromogenic substance, a pregnancy test reagent for pigs containing this as an essential component must be prepared in advance. By identifying the appropriate label that has been set, it is possible to easily identify whether or not the pregnant pig has the produced estrone in blood, saliva, or other body fluids. Pregnancy can be diagnosed relatively easily using body fluids, making it a diagnostic reagent for swine pregnancy with few errors.

Further, in order to solve the above-mentioned problems, 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 As a result, it is possible to easily detect the presence of estrone produced by pregnant pigs by aggregation of two or more molecules of ostium, and 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. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart showing the amino acid sequence of a branched, swine-produced ester-binding peptide. Embodiment of the Invention

In the present invention, 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.

When synthesizing peptides by solid phase synthesis, 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-α-9-fluorenylmethoxycarbonylated amino acid (Fmoc-amino acid) using a commercially available synthesizer controlled by a microcomputer On the other hand, the protecting peptide is appropriately removed to extend the required peptide chain in order. Incidentally, the manufacturers or sales companies of peptide synthesizers include Pharmacia Biotech, Perkin-Elma Japan, Aroka, and Shimadzu.

To produce a short peptide, a peptide is synthesized using Boc-Lys (Boc) or Fmoc-Lys (Fmoc) in an insoluble carrier such as cross-linked polystyrene. Here, Boc is a tertiary butyloxycarbonyl group, and Fmoc is a fluorenylmethoxycarbonyl group.

In order to grow the peptide, the Boc method or the Fmoc method, which is a method of synthesizing the peptide by a usual solid phase synthesis method, can be adopted. At this time, it is important to adjust the initial Lys content to about 0.1 mmo 1 / g because the number of reaction points increases.After the amino acid condensation reaction to grow peptide chains, 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). In addition, 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.

Further, in order to allow a predetermined number of peptides to be linked to the number of two or more lysines, a core in which lysines are linked in a linear or branched manner is used. In particular, for the number of lysines, n = l, 3, 7, and 15, 2, 4, 8, and 16 peptides can be connected in a branched manner.

Since two or more peptides are reacted in this way, the number of peptides reacting with each other increases, so that a stronger effect appears on a single peptide, and a test reagent that is sensitive to estrone can be obtained. .

For such a reason, 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).

For example, 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

-Crosslinking of antibodies, etc., can be performed, and the pork ester and the binding substance in the sample can be observed with the naked eye or a colorimeter using a peroxidase coloring reagent.

Furthermore, a method of cross-linking gold colloid ゃ latex at the N-terminus of SEQ ID NO: 1 or 2 and binding it to estrone in a swine sample, and visually observing the precipitate obtained when the method is adopted, can be adopted. .

As described above, 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. Example

Using an automatic peptide synthesizer (manufactured by Biosystems: ABI431A), the chemical synthesis method described in J. Am. Chem., 85, 2149 (1963) (solid phase synthesis by the 71-Boc method) Using B 0 P as a condensing agent in the method), and the method described in J.M.S (J) published in Biochemistry, Vol. 85, pp. 5409-5413 (issued in August 1998). According to the method of AM ESP (TAM), a branched peptide of MAP (Multiple antigen peptide) type was produced.

That is, a functional group was introduced into a polystyrene resin crosslinked with divinylbenzene. For resin (hydroxymethylphenylacetamide resin (also called PAM resin) or 4-methylbenzhydrylamine resin): Use an automatic peptide synthesizer that uses Boc-Lys (Boc). Using.

On an automated peptide synthesizer, 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. Next, after coupling 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. Thus, a branched peptide core in which 15 lysines (Lys) were linked was synthesized.

Next, 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. In solid-phase synthesis using an automatic peptide synthesizer (Boc method, 0.5 mmol scale), 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.

① Boc removal 50% TF A / dichloromethane 5 min vortex (stirring) Discharge the solution ""> 50% TFA / dichloromethane 30 min portex → discharge the solution

② Wash (discharge dichloromethane l min vortex solution) X 3 times-(DMF l min vortex → discharge solution) X 3 times

③ Condensed Boc-amino acids 1.5 mmol / DMF, BOP1 5 mmo1 / DMF, DIEA 2 mmol 40 min-> emission

洗浄 Washing (DMF l min vortex → drain the solution) x 3 times (draining the dichloromethane l min vortex solution) x 3 times.

Next, 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.

The obtained peptide was purified by high performance liquid chromatography (HPLC) using a reversed phase column. The conditions for HP LC were as follows: Wako Pure Chemical Industries, a column with an inner diameter of ø4.6 mm and a length of 25 Omm (Wakosi Le 2, 5C18 AR) packed with C18 silica gel with a particle size of 5 µm. The peptide was eluted using a 0.1% aqueous solution of tetrafluoroacetic acid (TFA) containing 5% acetonitrile (CH3CN) as the initial solvent.The acetonitrile concentration was 5 to 65%. Elution was performed for 30 minutes at a flow rate of 1.5 ml / min by temporarily setting a concentration gradient in the range.

As for the result of HPLC, a peak of absorbance was detected using an ultraviolet detector having a wavelength of 220 nm. The peak report at that time is as follows, and the amino acid sequence of the obtained branched peptide is shown in FIG.

Peak number Detection time (min) Area Height Concentration

1 1 3.6 46 6 52 3 1 5 7 1.0

2 1 5.5 47 7 6 0 7 6 3 0 7 6 2 9 8.5

3 2 3.0 0 2240 8 1 49 9 0.5 Dilute the branched peptide obtained as above at a predetermined ratio to prepare a pig pregnancy test reagent, and use it as follows. Inspection was carried out to confirm the effect.

[Pig pregnancy test experiment]

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. Dilute the ester-bonded short peptide prepared in Step 1 to 100 g / m1 with sterile distilled water and leave it at 37 for 24 hours. The presence or absence of aggregation due to the peptide conjugate was examined, and the results are shown in Table 1.

As is clear from the results in Table 1, 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.

Next, a pregnancy test experiment on pigs using saliva samples will be described.

Using an automatic peptide synthesizer (manufactured by Biosystems: ABI431A), the same chemical synthesis method as described in J. Am. Chem., 85, 2149 (1963) (by the t-Boc method) Using BOP as a condensing agent in the solid-phase synthesis method), the peptide represented by SEQ ID NO: 2 in the sequence listing in which two sets of peptides represented by SEQ ID NO: 1 are bound to one lysine ( A dimer of SEQ ID NO: 1) was produced.

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. A pregnancy confirmation experiment was performed using pig saliva as a sample.

Pregnancy was confirmed by ultrasonography, and 45 pregnant pigs and 3 non-pregnant sows and 5 boars were identified as test pigs as shown in Table 2 below. The saliva from the mouth of the pig before breakfast was collected with a cotton swab, and this was dissolved in 0.5 ml of a 9: 1 (volume ratio) mixture of chloroform: acetone in a glass container to disperse the estrone in the saliva. Extracted. — On the other hand, the peptide derivative prepared as described above is diluted with sterilized distilled water, and 10 ng (preferably in the range of 10 to 100 ng) of the peptide derivative is filtered through filter paper (ヮヅ F, 180F, manufactured by Toman). (5 mm width, 7 O mm length) and dried to prepare a thinner thin-layer chromatographic stationary phase (Chemco: silica gel thin-layer gel TLC) sandwiched with the filter paper. These were crimped and integrated into a chromatographic paper for inspection. Then, one end of the stationary phase was exposed from one end of the chromatographic paper for inspection, and the sample was absorbed (penetrated) from here.

Next, 1001 of the above-mentioned Estonia extract was absorbed from one end of the stationary phase, and after leaving it for several minutes, phosphate buffer 200 with pH 6.5 was further dropped and absorbed. Let me know.

A sample formed by determining the presence or absence of green fluorescent light based on the fraction formed 2 to 3 cm from one end of the filter paper of the inspection chromatograph paper and the part where the sample of the filter paper has not penetrated is used as a reference (blank). Were positive, non-colored samples were negative, and slight color development was judged as false positive. The results are shown in Table 2. Table 2

As is clear from the results in Table 2, the results of the pregnancy test of pigs using pig saliva as a sample showed that the correct answer rate was 100% for female pigs, and that the breeding was successful. It was found that it was possible to judge from the early pregnancy on and after the day. The invention's effect

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.

In the invention relating to the diagnostic reagent for swine pregnancy, 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. As a pregnancy diagnostic for pigs, it is possible to easily find the pregnancy-produced pig mouth bound to the label, and it can be diagnosed reliably using a relatively simple method. There are advantages.

In addition, in the invention of a pregnant swine-produced ester-binding peptide in which the C-terminal of a peptide comprising a specific amino acid sequence is linked to the N-terminus of a branched peptide, 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.

Claims

The scope of the claims

1. A peptide for a pig pregnancy test reagent comprising a peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and having a binding property to an ostium produced by a pregnant pig.

2. A pig consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing, which has binding properties to estrone produced by pregnant pigs, and has a peptide derivative having an N-terminal bound to a labeling substance as an active ingredient. Pregnancy test reagents.

3. Lysine (Lys) or a branched type lysine with two or more lysines linked to each end of a branch with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 Peptide.

4. A pig pregnancy test reagent comprising the pig pregnancy test reagent peptide according to claim 3 as an essential component. .