JP2011125268A - beta2 MICROGLOBULIN AND GENE THEREOF, ANTI-beta2 MICROGLOBULIN ANTIBODY, AND KIT AND METHOD FOR DIAGNOSING CAT NEPHROPATHY - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody that is specific to β2 microglobulin derived from cats, and further a method and kit capable of quickly and simply diagnosing cat nephropathy by using the same. <P>SOLUTION: There are disclosed a protein having a specific amino acid sequence and a structural gene encoding the protein, wherein the structural gene has a specific base sequence. There is disclosed an antibody capable of specifically binding to β2 microglobulin derived from cats, wherein the antibody is preferably produced by a cell line Mouse-Mouse hybridoma β<SB>2</SB>-m mAb1 (Receipt Number: FERM AP-21879), or a cell line Mouse-Mouse hybridoma β<SB>2</SB>-m mAb2 (Receipt Number: FERM AP-21880) using the protein as an antigen. There are disclosed a kit for diagnosing cat nephropathy including the antibody, and a method for diagnosing cat nephropathy using the antibody. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a β2 microglobulin derived from a cat and a gene encoding the same. The present invention also relates to an antibody against β2 microglobulin derived from a cat, a diagnostic kit for feline nephropathy using the same, and a diagnostic method.

  In recent years, with the declining birthrate, the number of households that keep pets is increasing. However, there are not a few cases where the pet is not kept according to the nature of the pet. In particular, as a result of uneven eating, pets cause adult pathological symptoms such as diabetes, and even pets are seen in veterinarians.

Under such circumstances, in recent years, businesses related to pet diagnosis are expanding. If a pet's nephropathy can be detected at an early stage, the veterinarian can give instructions on how to keep the pet, particularly how to eat, by the owner. In general, β2 microglobulin (β ₂ -m) is one of the markers of nephropathy.

For example, in the case of human origin, β2 microglobulin is produced in cells of the whole human body, and is secreted to the outside of the cell with a constant production amount with little influence on changes in the environment inside and outside the cell. However, there are reports that it is useful as an indicator for early diagnosis of diabetic nephropathy, etc. However, in the case of β2-microglobulin derived from cats, the amino acid sequence of the protein, rather than the presence of an antibody specific to the protein Even the current situation is not clear.

Journal of Veterinary Internal Medicine. 22 (5): 1111-1117, 2008

  The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide an antibody specific to feline-derived β2 microglobulin, and further to use it, the kidney of the cat It is to provide a method and a kit capable of quickly and easily diagnosing the disease.

  As a result of diligent research, the present inventor first identified a structural gene encoding β2 microglobulin among feline genes, expressed feline-derived β2 microglobulin from the structural gene, and analyzed its amino acid sequence. Furthermore, an antibody specific for cat-derived β2 microglobulin was prepared to complete the present invention. That is, the present invention is as follows.

The present invention provides a protein having the amino acid sequence represented by SEQ ID NO: 1.
The present invention also provides a structural gene encoding the protein of the present invention described above. The structural gene of the present invention preferably has the base sequence represented by SEQ ID NO: 2.

The present invention further provides an antibody that specifically binds to cat-derived β2 microglobulin. The antibody of the present invention can be prepared by using the above-described protein of the present invention as an antigen, cell line Mouse-Mouse hybridoma β ₂ -mmAb1 (reception number: FERM AP-21879) or cell line Mouse-Mouse hybridoma β ₂ -mmAb2 (reception). No .: FERM AP-21880).

The present invention also provides a kit for feline nephropathy diagnosis comprising the above-described antibody of the present invention.
The present invention also provides a method for diagnosing feline nephropathy using the antibody of the present invention described above.

  According to the present invention, feline nephropathy can be diagnosed much more quickly and simply than in the past.

FIG. 2 is a view showing the amino acid sequence of β ₂ -m derived from a cat in comparison with the amino acid sequences of β ₂ -m already known from humans, horses, cows, pigs, mice, monkeys, and rats. It is a figure which shows the base sequence of the cDNA of the β ₂ -m gene derived from a cat in comparison with the base sequences of β ₂ -m genes already known from humans, horses, cows, pigs, mice, monkeys and rats. . 6 is a photograph showing the experimental results of the specificity of antibodies A and B against cat native β ₂ -m in Experimental Example 3. FIG. It is a graph which shows the result of having quantified (beta) _2- m in the urine of one healthy cat and three chronic kidney disease cats using the antibodies A and B. 2 is an electrophoresis photograph showing the results of PCR of first-strand cDNA in Experimental Example 1. It is a figure which shows typically the positional relationship of cDNA which can be synthesize | combined with the kit used in Experimental example 1, and a primer. FIG. 7 (a) is a photograph showing the results of analysis of the cDNA amplified by the upstream primer 2 and Universal Primer A Mix by agarose gel electrophoresis, and FIG. 7 (b) is the result of electrophoresis after nested-PCR. It is a photograph which shows the result of analysis. It is a figure which shows typically the positional relationship of cDNA which can be synthesize | combined with the kit used in Experimental example 1, and a primer. 2 is an electrophoresis photograph showing the result of 5'RACE-PCR in Experimental Example 1. In Experimental example 2, it is a photograph which shows the result of having performed PCR, and having performed electrophoresis on the conditions which changed annealing temperature. In Experimental example 2, it is a photograph which shows the result of having carried out the agarose gel electrophoresis of pcDNA-F (beta) _2- m extracted from transformed Escherichia coli. In Experimental example 2, it is a figure which shows typically the sequence-analysis result of pcDNA-F (beta) _2- m. 6 is a photograph showing the results of SDS-PAGE in Experimental Example 2. It is a figure which shows typically the chromatogram for every urea concentration of the GST fusion protein solution in Experimental example 2. FIG. In Experimental example 2, it is a photograph which shows the result compared by SDS-PAGE about the ligand binding fraction of each urea concentration. In Experimental example 2, it is a photograph which shows the result of having reacted with PreScission Protease after adding DTT at each density | concentration to a ligand binding fraction, and dialyzing. It is a figure which shows typically the chromatogram of HPLC in Experimental example 2. FIG. It is a photograph which shows the result of SDS-PAGE about each fraction after HPLC.

According to the present invention, a protein having the amino acid sequence represented by SEQ ID NO: 1 is provided. The present inventor first identified a structural gene (referred to herein as a “β ₂ -m gene”) that encodes β2 microglobulin among feline genes, and the feline gene was identified by the β ₂ -m gene. The amino acid sequence of the derived β2 microglobulin (referred to herein as “β ₂ -m”) was also analyzed for the first time. The protein of the present invention having the amino acid sequence shown in SEQ ID NO: 1 is a cat-derived β2 microglobulin whose amino acid sequence has been specified for the first time by the present inventors.

Here, FIG. 1 shows the amino acid sequence of the protein of the present invention represented by SEQ ID NO: 1 (cat β ₂ -m), which is already known in human, horse, cow, pig, mouse, monkey and rat β. It is a figure shown in comparison with the amino acid sequence of _2- m. In FIG. 1, the portion surrounded by a square is an amino acid sequence common to each animal species. The total number of amino acids of the protein of the present invention represented by SEQ ID NO: 1 is 118, which is very close to that of 119 of humans, monkeys, mice and rats, and 118 of horses, cows and pigs. Although details will be described later in Experimental Example 2, the amino acid sequence of the protein of the present invention has an average homology with the amino acid sequence of β ₂ -m of other animal species of 72.8%, and other animal species (human, horse, bovine) The average homology of the amino acid sequence of β ₂ -m between (pig, mouse, monkey and rat) is 66.8%, and thus the protein of the present invention is considered to be β ₂ -m derived from cats.

The cat-derived β ₂ -m of the present invention can be suitably obtained by artificial synthesis. This time, the present inventor found for the first time the base sequence (base sequence represented by SEQ ID NO: 2) of the structural gene (β ₂ -m gene) derived from cat β ₂ -m. The present invention also provides a structural gene encoding feline-derived β2 microglobulin, and the structural gene preferably has the base sequence represented by SEQ ID NO: 2. That is, the structural gene of the present invention may contain a base sequence other than the above as an intron as long as it contains the base sequence represented by SEQ ID NO: 2 as an exon.

Here, FIG. 2 shows the structural gene of the present invention (cat β ₂ -m gene) represented by SEQ ID NO: 2 as previously known human, horse, cow, pig, mouse, monkey and rat β _2. It is a figure shown in comparison with the base sequence of the -m gene. In FIG. 2, the part enclosed by a square is a base sequence common to each animal species. nucleic length of beta ₂ -m gene, human, monkey, mouse and rat are 360 base, horse, whereas in cattle and swine was 357 base, cat of the present invention represented by SEQ ID NO: 2 beta ₂ - The length of the m gene was 357 base. Further, the homology between the base sequence of the β ₂ -m gene of the cat of the present invention represented by SEQ ID NO: 2 and the base sequence of the β ₂ -m gene of another animal species is 72.9% on average. The average homology of the base sequence of β ₂ -m gene among humans, horses, cows, pigs, mice, monkeys and rats) is 72.8%. Therefore, the structural gene of the present invention is β ₂ -m derived from cats. It is considered to be a gene.

The present invention also provides a novel antibody that specifically binds to cat-derived β ₂ -m. The present inventors, as described later in detail Experimental Example 3, was expressed beta ₂ -m from the Feline from beta ₂ -m gene from the Feline of the present invention described above, produce antibodies this as antigen Obtained cells were made. Such a cell line is novel, and the applicants have made a deposit at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of December 1, 2009 (reception number: FERM). AP-21879, FERM AP-21880).

The antibody of the present invention is preferably a cell line Mouse-Mouse hybridoma β ₂ -m mAb1 (reception number: FERM AP-21879) or a cell line Mouse-Mouse hybridoma β ₂ -m using the above-described protein of the present invention as an antigen. It was produced by mAb2 (reception number: FERM AP-21880). Here, FIG. 3 is a photograph showing experimental results in which the antibody of the present invention specifically binds to the native β ₂ -m of cats. Although details will be described later as Experimental Example 3, the antibody A shown in FIG. 3 is an monoclonal antibody of the isotype of the κ chain of IgG1 produced by the cell line Mouse-Mouse hybridoma β ₂ -mmAb1 (reception number: FERM AP-21879). Antibody B is an antibody of the IgG2b kappa chain isotype produced by the cell line Mouse-Mouse hybridoma β ₂ -mmAb2 (reception number: FERM AP-21880). As shown in FIG. 3, it can be seen that the antibody of the present invention can specifically bind to native β ₂ -m of cats. FIG. 4 is a graph showing the results of quantification of β ₂ -m in the urine of one healthy cat and three chronic kidney disease cats using the antibodies A and B of the present invention. . Although details will be described later in Experimental Example 3, it can be seen from FIG. 4 that antibodies A and B of the present invention hardly react to the urine of healthy cats, and all three cats to cats with chronic kidney disease. You can see that This suggests that a large amount of β ₂ -m is contained in the urine of cats with chronic kidney disease, and that the antibody of the present invention can be used for diagnosis of feline nephropathy.

The present invention further provides a diagnostic method and a diagnostic kit for feline nephropathy using the above-described antibody of the present invention. Since the antibody of the present invention can specifically bind to β ₂ -m, a marker for feline nephropathy, for example, whether or not the cat suffers from nephropathy using cat urine as a sample. Thus, it is possible to make a diagnosis more quickly and easily than in the past. In addition to the antibody of the present invention, the diagnostic kit of the present invention can contain, for example, wells, chromogenic substrate solutions, reaction stop solutions, washing solutions, standard solutions, and the like.

<Experimental example>
Hereinafter, although an example of an experiment is given and the present invention is explained in detail, the present invention is not limited to these.

<Experimental example 1: identification of β ₂ -m gene>
(1) Test animal In this experimental example, one 10-year-old male male Japanese cat that does not show any abnormality in blood biochemistry and urine biochemistry maintained at the laboratory animal facility was used. The cats were kept under conditions of 12 hours noon and 12 hours night in a cat cage, with free feeding and free drinking by feeding once a day.

(2) Extraction of Total RNA from Cat Leukocytes First, cat blood was collected from the external jugular vein of a test animal using an EDTA blood collection tube. The collected 5 ml of blood was transferred to a conical tube and centrifuged at 3000 × rpm for 5 minutes to separate the buffy coat (leukocyte layer). Next, Total RNA was extracted using QIAamp RNA Blood Kit (QIAGEN) according to the attached protocol. The obtained total RNA was stored at 4 ° C. until use.

Next, mRNA was separated and purified from total RNA using Oligotex ™ -dT30 Super mRNA Purification Kit (Takara Bio Inc.) according to the attached protocol. Specifically, 60 μl of total RNA was first mixed with 70 μl of 2 × Binding Buffer and 14 μl of Oligotex ™ -dT30, and then heated with a thermal climber (PC801, ASTEC) at 70 ° C. for 3 minutes. After warming, hybridization between mRNA and Oligotex ™ -dT30 Super was carried out by allowing to stand at room temperature for 10 minutes. Centrifuge the column containing the reaction solution at 15700 xg for 5 minutes, suspend in 350 μl Wash Buffer, transfer to the attached spin column set cup, centrifuge at 15700 xg for 30 seconds, and again with 350 μl Wash Buffer. After suspension, the mixture was centrifuged at 15700 × g for 30 seconds. Oligotex (trademark) -dT30 in the column was suspended in 30 μl of RNase free H ₂ O preheated to 70 ° C., and mRNA was eluted using the attached spin tube for spin column. This operation was repeated twice, and the resulting solution was used as an mRNA solution.

  Next, from the obtained mRNA solution, SMART ™ RACE cDNA Amplification Kit (Clontech) was used for sequencing the intermediate sequence and 3 ′ end, and CapFishing ™ was used for sequencing the 5 ′ end. First-strand cDNA was prepared using Full-length cDNA Premix Kit (Seegene) according to the attached protocol.

(3) Determination of the base sequence of the intermediate region of the cat-derived β ₂ -m gene Using Genetyx-Win version 7.1 (Software Development Co., Ltd.) in a region having higher homology than the base sequence of the animal species that has been clarified, A specific primer of the cat-derived β ₂ -m gene having the following base sequence was designed.

-Upstream primer 1: 5'-GGAAAGTCAAATAACCTGAA-3 '(SEQ ID NO: 3)
-Downstream primer 1: 5'-TCTCGATCCCACTTAACTATC-3 '(SEQ ID NO: 4)
Using the upstream primer 1 and the downstream primer 1 thus designed, first-strand cDNA was amplified by PCR using TaKaRa PCR Kit (Takara Bio Inc.) according to the attached protocol. Specifically, 1 μl of First-strand cDNA prepared in a 0.2 ml PCR tube, 2 μl of 10 × PCR Buffer, 2 μl of 25 mM MgCl ₂ , 2 μl of 8 mM dNTP, 5 units / ml AmpliTaq Gold (EC2.7.7.7, Applied Biosystems) 0.1 μl, 10 pmol / μl of gene-specific primersense primer and antisense primer 1 ml each was adjusted to 20 μl with distilled water (dH ₂ O), and the target cDNA was amplified using Mastercycler Gradient (Eppendorf) . Amplification was performed at 95 ° C for 10 minutes for 1 cycle, 95 ° C for 1 minute, 65 ° C for 1 minute, 72 ° C for 3 minutes for 35 cycles, and 72 ° C for 30 seconds for 1 cycle.

  To confirm the amplified cDNA fragment, agarose (SIGMA) was dissolved in 1 × TAE (40 mM Tris-HCl, 40 mM acetic acid, 1 mM EDTA, pH 8.0) at a rate of 1% or 2%. A sample for electrophoresis was prepared by mixing 18 μl of the PCR product and 2 μl of the loading buffer. 1 × TAE was used as an electrophoresis buffer, and electrophoresis was performed with an electrophoresis apparatus (Mupid-3, Cosmo Bio) at a constant voltage of 100 V for 30 minutes. After completion of electrophoresis, the agarose gel was stained with 100 ng / ml ethidium bromide (BIO-RAD) solution for 15 minutes and photographed using Epi-Light (FA500, Aisin Cosmos Laboratories). FIG. 5 is a photograph showing the results of analysis of the cDNA amplified by the PCR method using the upstream primer 1 and the downstream primer 1 by agarose gel electrophoresis. Although a band was confirmed around 240 bp, a smear-like migration image was observed. Therefore, after cloning this PCR product, the nucleotide sequence was analyzed.

Ligation was performed using TOPO ™ TA Cloning Kit (Invitrogen) according to the attached protocol. Specifically, first, in a 0.5 ml PCR tube, 2 μl of a PCR solution (Salt Solution 2M NaCl, 60 mM MgCl ₂ ) adjusted to 15 ng / μl, 1 μl of a vector plasmid (pCR2.1-TOPO), dH ₂ O 1 μl was added and reacted at 22.5 ° C. for 30 minutes.

  Next, transformation was performed using One Shot Chemical Transformation Kit (Invitrogen) according to the attached protocol. Specifically, first, 2 μl of the ligation reaction solution was added to a tube containing TOP 10 E. Coli, left on ice for 30 minutes, heated at 42 ° C. for 30 seconds, and immediately cooled on ice. Add 250 μl of SOC medium and incubate at 37 ° C. for 60 minutes, then inoculate 50 μl of LB agar plate medium coated with 20 μl of 100 mg / ml X-Gal (Takara Bio Inc.) per plate and evenly with a conical rod And then cultured at 37 ° C. for 18 hours. In addition, LB agar plate medium is added LB AGAR (GIBCO BRL (trademark)) 3.2mg 100ml ultrapure water, sterilized in an autoclave at 21 ° C for 15 minutes, then added ampicillin at a rate of 0.05mg / ml, It was made by hardening in a petri dish with a diameter of 9 cm.

Next, plasmid extraction was performed using QIAprep (trademark) Spin Miniprep Kit (QIAGEN) according to the attached protocol. TOP 10 E. Coli colonies in which insertion of the target DNA fragment was confirmed were taken with a sterile toothpick as much as possible, inoculated into 10 mM LB liquid medium, and cultured with shaking at 37 ° C. for 18 hours. Thereafter, the mixture was centrifuged at 15000 × g for 10 minutes, the supernatant was discarded, and a TOP 10 E.Coli pellet was obtained. This TOP 10 E.Coli was resuspended in 250 μl of TOP 10 E.Coli floating buffer (P1 Buffer) and transferred to a 1.5 μl tube. 250 μl of P2 Buffer (alkaline lysis buffer) was added, and the mixture was gently mixed by inversion to lyse, then neutralization buffer (N3 Buffer) was added within 5 minutes to stop lysis. After centrifugation at 10000 × g for 10 minutes, the supernatant was transferred to a spin column set in a 2 ml tube and centrifuged at 10000 × g for 1 minute. After discarding the supernatant, add 500 μl of propanol-containing guanidine hydrochloride buffer (PB Buffer) to the spin column, perform centrifugal washing at 10000 × g for 1 minute, and then add 750 μl of ethanol-containing desalting buffer (PE Buffer). The same centrifugal washing was performed. After the tube was replaced with a new one, 50 μl of dH ₂ O was dropped into the center of the spin column and centrifuged at 10000 × g for 1 minute to obtain a plasmid solution. The LB liquid medium used for TOP 10 E.Coli culture was added to 2 mg of LB (GIBCO BRL ™) in 100 ml of ultrapure water, sterilized in an autoclave at 21 ° C. for 15 minutes, and then 0.05 mg / ml of ampicillin. It was added at a rate of

(4) Analysis of nucleotide sequence in the whole cDNA
ABI PRISM (registered trademark) BigDye (registered trademark) Terminator Cycle Seqencing Ready Reaction Kit (Applied Biosystems) was used to prepare a sample for base sequence determination according to the attached protocol. In a 0.2 ml PCR tube, 1 μl of extracted plasmid adjusted to a concentration of 300 ng / μl, Terminator Ready Reaction Mix (fluorescein donor dye, 6-carboxy fluorescein) 8 μl, dH ₂ O 10.68 μl, 10 pmol / μl M13F and PCR reaction was performed using 0.32 μl of M13R primer and a thermal cycler. The PCR reaction was performed by repeating 25 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes.

  After completion of the reaction, the reaction solution was transferred to a 1.5 ml tube, 60 μl of 75% isopropyl alcohol was added, lightly mixed and allowed to stand for 20 minutes, and then centrifuged at 20000 × g for 20 minutes. All the supernatant was discarded, 250 μl of 75% isopropyl alcohol was added again, and the mixture was centrifuged at 20000 × g for 10 minutes, and then the water in the sediment was evaporated. The obtained PCR product was completely dissolved in 20 μl of Template Suspension Reagent (TSR, Applied Biosystems) and then denatured at 95 ° C. for 2 minutes. The base sequence was analyzed using ABI Prism 310 genetic Analyzer (Applied Biosystems). The obtained nucleotide sequence data was used to determine the nucleotide sequence of the cDNA using GENETYX-MAC ver.8.0 (Software Development) which is analysis software.

After cloning the PCR product, the nucleotide sequence was analyzed, and as a result, the nucleotide sequence shown in SEQ ID NO: 5 was obtained. When this sequence was compared with the base sequence of β ₂ -m cDNA of human, horse, cow, pig and mouse, and the homology of this sequence to each animal was analyzed, each homology was 75.3%, Since it was 80.0%, 77.1%, 79.6%, and 69.1%, it was determined to be an intermediate sequence of β ₂ -m cDNA.

(5) 3'-Rapid amplification of cDNA ends (RACE) -Neasted PCR method
A 3′-RACE-Neasted PCR method was performed using First-strand cDNA prepared from SMART ™ RACE cDNA Amplification Kit. Primer designs were made based on the obtained intermediate sequence, specific upstream primer 2 and upstream primer 3 each having the following base sequence. Furthermore, the downstream primer 3 for Nested-PCR was prepared in a region having a higher homology than the base sequences of human, mouse, rat, cow, pig, monkey and rat (FIG. 6).

-Upstream primer 2: 5'-GGGTTCCACCCACCAACAATTCAAAT-3 '(SEQ ID NO: 6)
-Upstream primer 3: 5'-TGGTCCACACCGAA-3 '(SEQ ID NO: 7)
-Downstream primer 3: 5'-GAAAATATGAAATACGTGTATT-3 '(SEQ ID NO: 8)
First PCR is performed using upstream primer 2 and Universal Primer A Mix (UPM: Clontech) (5'-AAGCAGTGGTATCAACGCAGAGG-3 '(SEQ ID NO: 9)), and then Second Nested-PCR is performed using upstream primer 3 And the downstream primer 3 combination. PCR conditions were 95 ° C for 10 minutes for 1 cycle, 95 ° C for 1 minute, 65 ° C for 1 minute, 72 ° C for 3 minutes for 35 cycles, and 72 ° C for 30 seconds for 1 cycle.

(6) Analysis of cDNA obtained by 3′RACE method and nested-PCR FIG. 7 (a) shows the result of analysis of cDNA amplified by upstream primer 2 and Universal Primer A Mix by agarose gel electrophoresis. It is a photograph. As shown in FIG. 7A, since a plurality of bands were confirmed in the electrophoresis image, nested-PCR was performed using the upstream primer 3 and the downstream primer 3 using this cDNA as a template. FIG. 7B is a photograph showing the result of electrophoresis analysis after nested-PCR. As a result, as shown in FIG. 7B, no band was confirmed in the upstream primer 2 and Universal Primer A Mix, and in the electrophoresis image of cDNA amplified using the upstream primer 3 and the downstream primer 3, Bands were observed near 220 bp and at positions below 100 bp. Thus, after cloning the PCR product confirmed at around 220 bp, the nucleotide sequence was analyzed, and as a result, it was found that the nucleotide sequence was shown in SEQ ID NO: 10. When the homology of each animal was analyzed with the nucleotide sequence of the obtained cDNA and various human, horse, bovine, porcine and mouse β ₂ -m cDNA sequences, the respective homology was 75.3%, 80.0% and 77.1%. 79.6% and 69.1%, and the 3 ′ end nucleotide sequence of the cat-derived β ₂ -m gene was revealed.

(7) 5'-Rapid amplification of cDNA ends (RACE) PCR method
5'-RACE PCR was performed using CapFishing (trademark) Full-length cDNA Premix Kit (Seegene). The mRNA concentrated and dried by the above-mentioned method is dissolved and mixed with 4 μl of 5 mM dNTP, 2 μl of 10 mM dT-adaptor and 4.5 μl of DEPC-treated water, followed by heating at 75 ° C. for 3 minutes and then rapidly cooling on ice for 2 minutes. After that, add 5 μl of RT Buffer 4 μl, 0.1 M DTT 1 μl, CapFishing (trademark) solution 1 μl, BSA (1 mg / ml) 2 μl, RNase inhibitor (40 IU / μl) 0.5 μl and Reverse transcriptase (200 IU / μl) 1 μl. In addition, it was incubated at 42 ° C. for 1 hour using a thermal cycler (ASTEC PC801). Subsequently, 3 ml of CapFishing ™ adapter and 0.3 ml of Reverse transcriptase (200 IU / μl) that had been pre-heated for 3 minutes in a constant temperature bath at 75 ° C. and then rapidly cooled on ice for 2 minutes were added to the reaction solution. It was again heated by a thermal cycler at 42 ° C for 30 minutes, 70 ° C for 15 minutes, and 94 ° C for 5 minutes. After heating at 94 ° C. for 5 minutes, the reaction solution was quenched on ice for 2 minutes, and then 180 μl of DEPC-treated water was added to obtain First-strand cDNA. The stored First-strand cDNA was subjected to PCR at an annealing temperature of 70 ° C. The downstream primer 4 used was designed to have the following base sequence based on the base sequence determined by analysis of the intermediate sequence. As the upstream primer, a 10 mM 5 ′ RACE (Rapid amplification of cDNA ends) primer of CapFishing (trademark) Full-length cDNA Premix Kit (Seegene) was used (FIG. 8).

-Downstream primer 4: 5'-GTGTGGACCAGAAGATAGAAAGTCC-3 '(SEQ ID NO: 11)
-5'RACE primer: 5'-GTCTACCAGGCATTCGCTTCAT-3 '(SEQ ID NO: 12)
After confirming the band that appeared in the vicinity of the theoretical length of the PCR product by agarose electrophoresis, the annealing temperature was adjusted to an ideal condition depending on the appearance state of the band. Cloning was performed using a DNA sample confirmed as a single band by agarose electrophoresis. After extracting plasmid-extracted DNA, the base sequence of the inserted DNA was determined. The base sequence was determined using an ABI PRISM BigDye (trademark) Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the attached protocol.

(8) Analysis of 5'RACE agarose gel electrophoresis images
The annealing temperature in PCR was set to 75 ° C., and 5′RACE method was performed. When agarose gel electrophoresis of the PCR product was performed, no band appeared. Therefore, the annealing temperature was lowered to 70 ° C., and the 5′RACE method was performed again. Here, FIG. 9 is a photograph showing the results of 5′RACE agarose gel electrophoresis, and lanes 1 to 3 are the results when the annealing temperatures were 75 ° C., 71 ° C., and 70 ° C., respectively. As shown in FIG. 9, although a band appeared in the vicinity of a target of about 350 bp but became smeared, when the annealing temperature was raised to 71 ° C., the band did not appear again. Therefore, the proper annealing temperature for the conditions of 5′RACE-PCR prepared using the 5′RACE primer and the downstream primer 3 was 70 ° C., and the base sequence was determined after cloning. As a result of analyzing the base sequence, it was found to be the base sequence shown in SEQ ID NO: 13. As a result of analyzing the homology of the obtained base sequence and the base sequence of β ₂ -m cDNA of human, horse, cow, pig, mouse, monkey and rat, the respective homology was 74.4%, 78.3%, They were 78.3%, 78.3%, 69.3%, 72.7% and 69.3%. Therefore, this sequence was determined as a 5 ′ end sequence.

(9) Analysis of the base sequence in the entire cDNA obtained The entire sequence was constructed based on the base sequence obtained as described above (SEQ ID NO: 2). Figure 2 is the nucleotide sequence of the cDNA of beta ₂ -m gene resulting cats, humans already known, horses, cows, pigs, mice, and the base sequence of the beta ₂ -m gene monkey and rat compared It is a figure shown. In the drawing, base sequence portions that are common to each other are shown by being surrounded by a square. The cDNA length was 360 bases in humans, monkeys, mice, and rats, and 357 bases in horses, cows, and pigs, whereas in the feline cDNA shown in SEQ ID NO: 2, the length was It was 357 base. In addition, the average homology of β ₂ -m cDNA nucleotide sequence among other animal species (human, horse, cow, pig, mouse, monkey and rat) is 72.9% on average (Table 1). The homology between the nucleotide sequence and other animal species was 73.4%, 76.7%, 74.5%, 76.5%, 67.3%, 71.2%, and 68.1%, and the average homology was 72.5% ( Table 2). This revealed that the obtained base sequence was the cat β ₂ -m gene.

<Experimental example 2: Synthesis of cat-derived β ₂ -m>
(1) translating the amino acid sequence from the beta ₂ -m gene nucleotide sequences of feline origin obtained in analyzing experimental example 1 cat from beta ₂ -m amino acid sequence (SEQ ID NO: 2), from the Feline beta ₂ - The amino acid sequence of m (SEQ ID NO: 1) was analyzed. FIG. 1 shows the amino acid sequence of the obtained cat β ₂ -m in comparison with the amino acid sequences of β ₂ -m of already known humans, horses, cows, pigs, mice, monkeys and rats. It is. The total number of amino acids of the protein of the present invention represented by SEQ ID NO: 1 was 118, which was very close to 119 of humans, monkeys, mice and rats, and 118 of horses, cows and pigs. Moreover, the average homology of the amino acid sequence of β ₂ -m among other animal species (human, horse, cow, pig, mouse, monkey, and rat) was 72.8% (Table 3). Moreover, when the sequence obtained by translating the nucleotide sequence obtained this time into amino acids was compared with the β ₂ -m amino acid sequences of other animal species, the respective homology was 65.3%, 72.6%, 68.4%, 74.4%. 60.8%, 63.6% and 62.7% with an average homology of 66.8% (Table 4). Therefore, it was revealed that the obtained amino acid sequence was β ₂ -m derived from cats.

(2) Primer design Primers were designed from the base sequence of the obtained feline β ₂ -m mRNA using Genetyx-Win version 5.1 (Software Development Co., Ltd.). Since pGEX-6P-1 (GE Healthcare Bioscience) is used as a vector, Bam HI is added to the 5 ′ end of the upstream primer and Sal I restriction enzyme recognition sequence is added to the 5 ′ end of the downstream primer. A base sequence was prepared. N represents T, A, C, or G.

-Upstream primer: 5'-NNNGGATCCGTCCAGCATTCCAAAGGTTCAGGT-3 '(SEQ ID NO: 14)
-Upstream primer: 5'-NNNGTCGACTTACATGTCTCGATCCCACTTAACGACCTT-3 '(SEQ ID NO: 15)
(3) PCR method Mix 4 μl of the above first strand cDNA, 12.5 μl of GoTaqR Green Master Mix (Promega), 1 μl of upstream primer, 1 μl of downstream primer and 6.5 μl of RNase free H ₂ O in a PCR tube. PCR was performed. The PCR conditions were as follows: 95 ° C for 2 minutes, 95 ° C for 45 seconds, primer pair annealing temperature for 45 seconds, 72 ° C for 1 minute 35 cycles, then 72 ° C for 7 minutes. In addition, the annealing temperature was examined in order to find the optimal conditions for PCR. In RT-PCR performed at an annealing temperature of 77.5 ° C., a band of about 50 bp appeared in addition to a band of about 300 bp which seems to be the target β ₂ -m cDNA. Here, FIG. 10 is a photograph showing the results of performing PCR under conditions with different annealing temperatures and performing electrophoresis on a 2% agarose gel. Lanes 1 to 3 show annealing temperatures of 77.5 ° C., 80 ° C., respectively. In the case of 85 ° C., lanes 4 and 5 are when the amount of primer added is changed. This approximately 50 bp band decreased as the annealing temperature increased to 77.5 ° C, 80 ° C and 85 ° C, but did not disappear. Therefore, when the annealing temperature was set to 85 ° C. and the amount of primer added was changed, and the amount of primer added was reduced to half in lane 5, the band of about 50 bp was reduced and the band considered to be a β ₂ -m cDNA band. A PCR product with a nearly single band of 300 bp was obtained and used as the cDNA inserted into the vector plasmid.

(4) DNA extraction from gel
DNA extraction from the gel was performed using QIAquick Gel Extraction Kit (QIAGEN) according to the attached protocol. The target DNA band of the agarose gel was cut out and the weight of the gel was measured. The excised gel was added with 3 times the amount of QG buffer and heated in a 50 ° C thermostat (TR-2A, ASONE) for 10 minutes to completely dissolve the gel, and then the same amount of isopropanol as the gel was added. In addition, well mixed. The DNA solution was added to a 2 ml collection tube with a column attached to the kit, and centrifuged at room temperature at 13400 × g for 1 minute. Then, after discarding the filtrate in the collection tube, 0.75 ml of PE buffer was added to the column again, and after washing by centrifugation at 15700 × g for 1 minute at room temperature, the filtrate was removed and centrifuged for another 1 minute. . Then, set the column in a new 1.5 ml microtube, add 50 μl of EB buffer, leave it at room temperature for 1 minute, collect the extract by centrifugation at 15700 xg for 1 minute, and add the resulting solution to the solution. A DNA extraction solution was used.

(5) Concentration of PCR products
The DNA extraction solution was mixed with phenol in an equal amount, and centrifuged at 15700 × g for 5 minutes, and then the aqueous layer containing the nucleic acid was separated. The aqueous layer was mixed with an equal amount of chloroform, centrifuged at 15700 × g for 5 minutes, and the supernatant was separated. Next, the separated solution was added with 2.5 times volume of 100% ethanol and allowed to stand at −80 ° C. for 30 minutes, then centrifuged at 15700 × g for 5 minutes, and the supernatant was removed to obtain a precipitate. 70% ethanol was added to the precipitate, and after centrifugation at 15700 × g for 5 minutes, the supernatant was removed to obtain a concentrated sample of the PCR product.

(6) Preparation of β ₂ -m cDNA integration vector and E. coli transformation method
Concentrated samples of PCR products were 5 μl of BamHI (Takara Bio Inc.), 5 μl of SalI (Takara Bio Inc.), 5 μl of H.Buffer (500 mM Tris-HCl, pH 7.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1000 mM NaCl) and RNase free H ₂ O was mixed with 35 μl. In addition, 5 μl (2.5 μg) of pGEX6P-1 was mixed with 5 μl of BamHI, 5 μl of SalI, 5 μl of H.Buffer, and 30 μl of RNase free H ₂ O. Each solution was subjected to restriction enzyme treatment by incubating overnight at 37 ° C., followed by agarose gel electrophoresis, and extraction of each DNA band using the QIAquick Gel Extraction Kit. Ligation was performed using DNA Ligation Kit (Takara Bio Inc.). Specifically, 5 μl of Ligation Mix, 1 μl of restriction enzyme-treated β ₂ -m cDNA solution and 4 μl of pGEX6P-1 were mixed and allowed to stand at 16 ° C. overnight. Then, β ₂ -m cDNA was transfected using this reaction solution. Add 2.5 μl of the reaction solution to 25 μl of E. coli JM109 Competent Cells (Takara Bio Inc.), let stand on ice for 30 minutes, then apply heat-shock for 45 seconds in a 42 ° C constant temperature bath, and immediately ice for 2 minutes. After cooling, 250 μl of SOC medium (2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO ₄ , 10 mM MgCl ₂ , 20 mM glucose) was gently added and incubated at 37 ° C. for 1 hour. 100 μl of β ₂ -m cDNA transfection solution was applied to ampicillin-added LB medium, allowed to stand overnight at 37 ° C, colonies were picked, mixed with 1.2 ml of ampicillin-added LB liquid medium, and then overnight at 37 ° C. Cultured. The cultured liquid medium was centrifuged at 13400 × g for 1 minute, the supernatant was completely removed, and plasmid extraction was performed from the resulting precipitate.

(7) Plasmid extraction
Plasmid extraction was performed using QIAPrep Spin Mini Kit 50 (QIAGEN) according to the attached protocol. Specifically, first, the above-obtained sediment was dissolved in 250 μl of buffer P1, 250 μl of buffer P2 was added, and then lysed by gently inverting and mixing. The lysis reaction was stopped by adding 350 μl of neutralized N3 buffer, followed by centrifugation at 15700 × g for 10 minutes, and the supernatant was added to a collection tube with a column attached. After centrifuging the column at 5900 × g for 1 minute, the filtrate was removed and 500 μl of Binding Buffer was added. Subsequently, after centrifugal washing at 9300 × g for 1 minute, the filtrate was removed, 750 μl of ethanol-containing desalting buffer was added, and after centrifugal washing at 9300 × g for 1 minute, it was transferred to a new tube. 50 μl of Elution Buffer was added to the column membrane and centrifuged at 9300 × g for 1 minute to obtain a plasmid extraction solution. This plasmid was designated pcDNA-F β ₂ -m and confirmed by agarose gel electrophoresis. FIG. 11 is a photograph showing the results of agarose gel electrophoresis of pcDNA-F β ₂ -m extracted from transformed E. coli. In FIG. 11, lanes 1 and 2 are a plasmid (pGEX6p-1) and β ₂ -m cDNA treated with restriction enzymes (BamHI and Sal1), respectively, and lane 3 is pcDNA-F β ₂ -m and lane 4 Shows the results for a sample obtained by treating pcDNA-F β ₂ -m with restriction enzymes (BamHI and Sal1). As shown in FIG. 11, a thick band was confirmed at about 3000 bp, and a thin band was observed at about 8000 bp, about 5000 bp, and about 2000 bp. Further, in lane 4 in which a sample obtained by subjecting the extracted pcDNA-F β ₂ -m to restriction enzyme treatment with BamH1 and Sal1 was run, bands of about 5000 bp and about 300 bp were confirmed. These two bands of about 5000 bp and about 300 bp were bands having substantially the same molecular weight as compared with the electrophoresis results of β ₂ -m cDNA and pGEX6P-1 before transduction. The success or failure of pcDNA-F β ₂ -m subcloning was performed by sequence analysis using T7 primer and Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems). FIG. 12 is a diagram schematically showing the result of sequence analysis of pcDNA-F β- ₂ m. From the results shown in FIG. 12, it was confirmed that the base sequence of the incorporated β ₂ -m cDNA was correctly incorporated into the vector.

(8) Confirmation of GST fusion protein expression After culturing the transfected E. coli in LB medium at 37 ° C overnight, mix 100 µl with 20 µl of Isopropl-β-D-thiogalactopyranoside (IPTG: 0.1 mM) at 37 ° C. The culture was shaken for about 2 hours (BR40-LF, TAITEC). The E. coli solution after shaking culture is centrifuged at 15700 × g for 1 minute, and then the supernatant is removed. The solubilized solution (50 μM Tris-HCl 50 μl, 1 × RIPA Lysis Buffer (Up State) 100 μl, Protein Inhibitor 140 μl) , 30 μl of H ₂ O (710 μl), solubilized, centrifuged at 15700 × g for 5 minutes, and separated into supernatant and sediment. Add 30 μl of 2 × SB solution (2% SDS, 40% Glycerol, 0.6% BPB, 25 mM Tris-HCl Buffer (pH 6.8, 20 ° C.)) and 1 μl of 2ME to 30 μl of the supernatant, and warm at 95 ° C. for 3 minutes. did. To the sediment, 20 μl of SB solution was added, crushed with an ultrasonic crusher (UR-20P, TOMY SEIKO CO, LTD) for 5 seconds, and then heated at 95 ° C. for 3 minutes. Thereafter, the supernatant and sediment were confirmed by SDS-PAGE for the expression of GST fusion protein and the solubility of GST fusion protein in Escherichia coli. E. coli obtained after GST fusion protein induced expression was sonicated, and the supernatant and sediment obtained by centrifugation were electrophoresed on SDS-PAGE. FIG. 13 is a photograph showing the results of SDS-PAGE. Lane 1 shows the results for the supernatant, and Lane 2 shows the results for the sediment. The molecular weight of the GST fusion protein was about 37 kDa, and no significant band could be confirmed in the supernatant, but a clear thick band was confirmed in the sediment. Therefore, it was confirmed that the GST fusion protein was expressed in the insoluble fraction.

(9) SDS-PAGE method Based on the Laemmli method using compact PAGE (AE-7300, ATTO), SDS-PAGE was performed with the following modifications. Specifically, the composition of the separation gel was 15% acrylamide, 0.2% N, N-methylene-bis-acrylamide, 0.1% SDS, 375 mM Tris-HCl buffer (pH 8.8, 20 ° C.). The gel was prepared using a 2/4 series gel preparation device (AE-7344, ATTO). The composition of the electrode buffer was 0.1% SDS, 129 mM glycine, 25 mM Tris (pH 8.3, 20 ° C.). The composition of the sample for electrophoresis (SB) was 1% SDS, 20% glycerol, 0.3% BPB, 12.5 mM Tris-HCl Buffer (pH 6.8, 20 ° C.). In addition, a prestained SDS-PAGE standard (Broad) marker (BIO-RAD) or an SDS-PAGE standard (Broad) marker (BIO-RAD) was used as a marker. The electrophoresis was terminated when the Tris-Gly / PAGE High mode was run for 30 minutes and then the Tris-Gly / PAGE Low mode was set, and the lower ion interface was moved to a position 1 to 2 mm above the bottom of the gel. The gel after SDS-PAGE was subjected to a silver staining method based on the Oakley method. That is, the gel was fixed with 30% ethanol and 10% acetic acid solution, washed, and immersed twice in 20% ethanol for 5 minutes. After removing 20% ethanol, it was reacted for 4 minutes with a 5% glutaraldehyde solution, washed with pure water, and then immersed twice in 20% ethanol for 4 minutes. Thereafter, it was washed with pure water, reacted for 5 minutes with an ammoniacal silver nitrate solution, washed with pure water, and then developed with 0.005% citric acid and 0.019% formaldehyde solution. The gel after color development confirmation was fixed with 20% ethanol and 10% acetic acid solution for 5 minutes, immersed in 20% ethanol twice for 5 minutes, and a photograph was taken. All silver staining methods were performed under light-shielding conditions.

(10) Induction and isolation of GST fusion protein expression
Escherichia coli in which the expression of the GST fusion protein was confirmed was applied to ampicillin-added LB agar medium, colonies were picked, added to 3 ml of ampicillin-added LB liquid medium, and cultured overnight at 37 ° C. with shaking. Subsequently, 3 ml of the culture solution was added to 250 ml of ampicillin-added LB liquid medium, and after shaking culture at 37 ° C. for about 150 minutes, 2.5 ml of 0.1 mM IPTG was added, and GST fusion protein cultured at 37 ° C. for about 2 hours. The protein composition after protein expression was analyzed. The GST fusion protein expression after induction of the culture solution centrifuged sediment for 15 minutes at 6000 × g, 0.5mM EDTA, 0.4M NaCl in 20ml, 5mM MgCl _2, 5% glycerol, 0.5mM phenylmethylsulfonyl fluoride (PMSF), 1mM dithiothreitol (DTT) and 1 mg / ml lysozyme-added 50 mM Tris-HCl (pH 8.0) were suspended, allowed to stand at 4 ° C. for 1 hour, and then freeze-thawed twice. Subsequently, 0.5% Nonidet P-40 was added, and after crushing for 20 seconds × 5 times with an ultrasonic crusher, the mixture was centrifuged at 9300 × g for 20 minutes, and the supernatant was removed to obtain a precipitate. The resulting precipitate was resuspended in 10 ml of 8M Urea, 0.5 mM DTT added Phosphate buffer saline (PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ PO ₄ , 1.8 mM KH ₂ PO ₄ , pH 7.3) 4 The mixture was allowed to stand at 1 ° C. for 1 hour and centrifuged at 9000 × g for 20 minutes to obtain a supernatant. This supernatant was used as a GST fusion protein solution.

(11) Affinity chromatography method
A peristaltic pump (SJ-1211L, ATTO) was added to a GSTrap HP column column (GE Healthcare Bioscience) equilibrated with 0.5 M Urea-added PBS with 20 ml of GST fusion protein solution at a flow rate of 0.3 ml / min. In addition, about the GST fusion protein solution added to a column, the adsorption amount of GST fusion protein to a column by the difference in urea concentration was compared. The column was washed with 0.5 M Urea-added PBS, and eluted with 10 mM reduced glutathione and 1 M Urea-added 50 mM Tris-HCl (pH 8.0). The absorbance of the GST fusion protein eluate was monitored at an absorbance wavelength of 220 nm using an ultraviolet absorbance monitor (AC-5100L, ATTO) and recorded with a recorder (R-01A, RIKADENKI). 2 ml of the obtained GST fusion protein eluate was mixed with DTT so that the concentration was 1 mM, and then placed in a dialysis membrane (UC30-32-100, Sanko Junyaku Co., Ltd.) with a molecular weight of 13 kDa, 150 mM NaCl, Dialysis was performed for about 6 hours using 2 L of 1 mM EDTA and 50 mM Tris-HCl (pH 7.5). The amount of GST fusion protein bound to HiTrap affinity column was compared by the difference in urea concentration (2M, 1M and 0.5M). The chromatograms of the GST fusion protein solution at each urea concentration are shown in FIGS. 14 (a), (b) and (c), respectively. FIGS. 14A, 14B, and 14C show chromatograms using protein samples with urea concentrations of 2M, 1M, and 0.5M, respectively. In each chromatogram, the absorbance change due to the addition of the sample was observed in the first half, and the absorbance decreased by the addition of the washing buffer. Thereafter, a sharp peak was confirmed by the addition of elution buffer, but an increase in absorbance was observed as the urea concentration decreased, and the sharpest fraction was observed at a urea concentration of 0.5M. FIG. 15 shows the results of comparison by SDS-PAGE of the ligand-binding fractions at each urea concentration. In FIG. 15, lane 1 is the ligand binding fraction obtained in FIG. 14 (c), lane 2 is the ligand binding fraction obtained in FIG. 14 (b), and lane 3 is obtained in FIG. 14 (a). Results for the ligand binding fraction are shown. As shown in FIG. 15, a decrease in the amount of GST fusion protein bound was confirmed as the urea concentration increased, and the ligand-binding fraction of affinity chromatography performed at a urea concentration of 0.5 M (obtained in FIG. 14 (c)). (Hereinafter referred to as “C4 fraction”) was used in the following experiments.

(12) Protease treatment
After dialysis, GST fusion protein eluate was quantified using DC Protein Assay (Bio-Rad), and 1 μl of PreScission Protease (GE Healthcare Bioscience) was added to 200 μg of protein and mixed. The sample was reacted at 4 ° C. for 6 hours or more to prepare a sample for high performance liquid chromatography (HPLC). The effects of dialysis and DTT on the PreScission Protease reaction were investigated. In addition, the effect of DTT concentration on PreScission Protease activity was examined using C4 fraction. FIG. 16 shows the results obtained by adding DTT to the C4 fraction to final concentrations of 1 mM, 2.5 mM, and 5 mM and reacting with PreScission Protease after dialysis. In FIG. 16, lane 1 shows a case where 1 mM DTT is added, lane 2 shows a case where 2.5 mM DTT is added, and lane 3 shows a case where 5 mM DTT is added. Although eventually the concentration beta ₂ -m band of approximately 11kDa is also in DDT addition of was observed, in 1mM of DTT addition disappearance of the band of approximately 15 kDa, the DTT addition of 2.5mM and 5 mM, beta ₂ -m bands In addition, a band of about 13 kDa was confirmed. From the above results, the high-performance liquid chromatography method (HPLC), which is the next purification step, used a sample that had been treated with PreScission Protease after dialysis of the C4 fraction added with 1 mM DDT.

(13) HPLC method
HPLC system includes system controller (SCL-10A VP, Shimadzu), liquid feeding unit (LC-10AD VP, Shimadzu), ultraviolet partial altimeter (SPD-10A VP, Shimadzu), column oven (CTO-10A VP, Shimadzu) And a degassing unit (DGU-14A, Shimadzu), and MightysilRP-18 GP250-4.6 (Cat. No. 25415-96, Kanto Chemical) was used as the column. The HPLC separation conditions were as follows: the mobile phase flow rate was 1 ml / min, the sample addition amount was 400 μl, equilibrated with 0.1% Trifluoroacetic Acid (TFA) solution, and the column was used with 0.1% TFA-added acetonitrile solution, and the acetonitrile concentration was 0-80. % Liner gradient. The eluate was monitored for absorbance at an absorption wavelength of 220 nm, the detected peak was collected, centrifuged for 1 hour in a concentration centrifuge (CC-181, TOMY), and then freeze-dried (FDU-540, EYELA) and stored at -20 ° C. Moreover, the protein of each eluted fraction was analyzed by SDS-PAGE. FIG. 17 is a diagram schematically showing an HPLC chromatogram. The C4 fraction after protease treatment was eluted mainly as 5 fractions. These five fractions were C4a, C4b, C4c, C4d and C4e in the order of elution and analyzed by SDS-PAGE. FIG. 18 is a photograph showing the results of SDS-PAGE for each fraction after HPLC. As shown in FIG. 18, no band was confirmed in the C4a fraction, about 11 kDa bands in the C4b fraction, about 11 kDa, about 25 kDa, and about 27 kDa bands in the C4c fraction, and about 11 kDa in the C4d fraction. A band of about 27 kDa was confirmed, and a band of about 27 kDa was confirmed in the C4e fraction. The protein considered to be the target β ₂ -m was detected as a single band in the C4b fraction. The acetonitrile concentration at the elution of the C4b fraction was 39.5%. This eluate was concentrated and centrifuged as recombinant cat β ₂ -m, stored at −80 ° C. after lyophilization.

<Experimental Example 3: Production of antibody-producing hybridoma and anti-rFeβ ₂ -m antibody>
In preparing a monoclonal antibody using the protein synthesized in Experimental Example 2 as an antigen of a recombinant cat β ₂ -m (rFeβ ₂ -m), an antibody-producing hybridoma was first prepared.

(1) Preparation of antibody-producing hybridoma (1-1) Immunization The immunization was carried out by injecting purified rFeβ ₂ -m as an antigen subcutaneously into the hindlimb paws (footpad) of Balb / c mice. Immunization is performed 4 times at 5-day intervals, and from the first to the third immunization, 100 μl (1 mg / ml) of antigen solution and 200 μl of antigen solution (50 μg / foot) emulsified by mixing an equal amount of adjuvant In the final immunization, only 20 μl (10 μg / foot) of the antigen solution was used. Adjuvant Complete Freund (Wako Pure Chemical Industries, Ltd.) was used as the adjuvant for the first immunization, and Adjuvant Incomplete Freund (Wako Pure Chemical Industries, Ltd.) was used for the second to third immunizations.

(1-2) Cell fusion Three days after the final immunization, popliteal lymph nodes were removed, lymphocytes were collected, and then cell fusion was performed using GenomONE-CF (Ishihara Sangyo Co., Ltd.). As myeloma cells, P3X63-Ag8.653 (Dainippon Sumitomo Pharma Co., Ltd.) was used. The fusion method was performed according to the attached protocol. Specifically, first, lymphocytes and myeloma cells were mixed at a cell number ratio of 5: 1, centrifuged at 1000 rpm at 4 ° C. for 5 minutes, and then the supernatant was removed. Thereto was added 1 ml of ice-cold fusion buffer per 10 ⁸ cells of lymphocytes and suspended uniformly, and then 25 μl of ice-cooled HVJ-Envelope suspension was added per ml of cell mixture. The cell suspension was allowed to stand on ice for 5 minutes, then centrifuged at 1000 rpm and 4 ° C. for 5 minutes, and incubated at 37 ° C. for 15 minutes with the cells pelleted without removing the supernatant.

After completion of the incubation, 50 ml of a growth medium heated to 37 ° C. was added per 10 ⁸ lymphocytes, suspended, and then seeded in a 96-well plate (96 Well Cell Culture Plate: Greiner bio-one) at 100 μl / well. As growth medium, RPMI1640 (Invitrogen), penicillin G (PG; Meiji Pharmaceutical Co., Ltd.) 100,000 IU / ml, streptomycin (SM; Meiji Pharmaceutical Co., Ltd.) 100 mg / ml, 7.5% Bri Clone (IL-6, human) No. BR-001, Dainippon Sumitomo Pharma Co., Ltd.), 10% inactivated fetal bovine serum (FBS; Nichirei Co., Ltd.) added, and gently added and suspended. Operated. After culturing for 24 hours, the culture medium was replaced with a HAT medium in which 2% HAT (Invitrogen) was added to the above growth medium.

(2) Screening for antibody-producing hybridomas The resulting hybridomas were subjected to a primary screening using an ELISA method one week after cell fusion. As a result, only those hybridomas in which the reaction was positive were detected using Western blotting. Confirmed by subsequent screening.

(2-1) Primary screening
Primary screening of antibody-producing hybridomas was performed using an ELISA method using rFeβ ₂ -m as an antigen. As an ELISA plate, 96 well ELISA Microplate (Greiner bio-one) was used. In addition, an automatic washer (Auto Mini Washer AMW-8, Biotech Co., Ltd.) was used to wash the plate, and PBS (1.37M NaCl, 27 mM KCl, 100 mM Na ₂ HPO ₄ , 18 mM KH ₂ PO ₄ , pH 7.4, 25 ° C.) was used. As a solid phase, rFeβ ₂ -m adjusted to 3 μg / ml with PBS was added to the plate at 50 μl / well and reacted overnight at 4 ° C. After the solid-phase reaction was completed, the antigen solution on the plate was discarded, and PBS supplemented with 0.5% Bovine Serum Albumin (BSA; Wako Pure Chemical Industries, Ltd.) was added as a blocking solution at 150 μl / well and reacted at 37 ° C. for 60 minutes. . After completion of the blocking reaction, the plate was washed once, and the culture supernatant of each hybridoma culture was added at 50 μl / well as a primary antibody, and reacted at 37 ° C. for 60 minutes. After completion of the primary antibody reaction, the plate was washed once, and a peroxidase-labeled anti-mouse IgG antibody (SIGMA-ALDRICH) diluted 1000-fold with PBS supplemented with 0.1% BSA as a secondary antibody was added at 50 μl / well at 37 ° C. For 60 minutes. After completion of the secondary antibody reaction, the plate was washed 3 times, and PBS containing 0.04% o-phenylenediamine and 0.04% H ₂ O ₂ was added as a substrate solution at 150 μl / well, and room temperature was kept for 30 to 60 minutes under light shielding. Reacted. After completion of the substrate reaction, 3M H ₂ SO ₄ was added as a reaction stop solution at 50 μl / well, shaken for 1 minute, and the absorbance at a wavelength of 490 nm was measured with a Microplate Reader (Model 550, BIO-RAD). The positive well cells having high absorbance were transferred to a 24-well plate (24 Well Cell Culture Plate; Greiner bio-one) and cultured.

(2-2) Secondary screening
This was confirmed by Western blotting using rFeβ ₂ -m as an antigen, and secondary screening of antibody-producing hybridomas was performed. Based on Lowry's method, the protein was quantified by measuring the absorbance at a wavelength of 655 nm with a Microplate Reader using a DC Protein Assay Kit (BIO-RAD). A calibration curve was prepared using BSA. The Western blotting method was performed as follows based on the method of Towbin et al. A polyvinylidene difluoride (PVDF) film (BIO-RAD) was used as the transfer film. The PVDF membrane was infiltrated with 100% methanol for 10 seconds and further with a transfer electrode buffer (25 mM Tris-HCl (pH 8.3, 20 ° C.), 192 mM glycine, 5% methanol) for 30 minutes and subjected to electrophoresis. In assembling the transfer device, a filter paper (BIO-RAD), a PVDF membrane, a gel after completion of SDS-PAGE, and a filter paper were layered in order from the bottom on the anode electrode plate, and the cathode electrode plate was fixed thereon. The filter paper was previously immersed in the electrode buffer for 2 to 3 minutes. The transfer condition was a constant current of 1.9 mA / cm ² for 60 minutes. After the transfer, the PVDF membrane was subjected to blocking operation by adding 0.5% BSA to 10 mM Tris-HCl (pH 7.5, 20 ° C.), 140 mM NaCl, 0.01% Tween 20 (TBST) and shaking at room temperature for 60 minutes. After the blocking, the cells were shaken and washed twice with TBST for 5 minutes, and the cell culture supernatant was used as the primary antibody, and the reaction was shaken at room temperature for 90 minutes. After completion of the primary antibody reaction, the mixture was washed twice with TBST for 5 minutes, and then peroxidase-labeled anti-mouse IgG antibody diluted 1000 times with TBST was shaken at room temperature for 60 minutes. After completion of the secondary antibody reaction, shake and wash twice with TBST for 5 minutes and use 0.06% 3,3-diaminobenzidine tetra-hydrochloride, 0.03% H ₂ O ₂ and 50 mM Tris-HCl (pH 7.6, 20 ° C.) as a substrate. It was used as a reaction solution and allowed to react for 1 to 5 minutes. After completion of the substrate reaction, the reaction was stopped by washing with water, and then dried and stored. Hybridomas that showed positive reaction were cloned by the limiting dilution method described later.

(3) Cloning The limiting dilution method was used for the cloning of the hybridoma. Specifically, the hybridoma after screening was diluted with HAT medium so as to be 2 cells / 100 μl, and seeded in a 96-well plate at 100 μl / well. The hybridoma was expanded to a 24-well plate when it became semi-confluent, cultured again until it became semi-confluent, and then confirmed by Western blotting using rFeβ ₂ -m as an antigen as in the secondary screening. This cloning operation was performed twice. In addition, in order to prevent the antibody-producing ability from being reduced by subculturing the hybridoma for a long period of time, it was stored using a cell cryopreservation solution (Cell Banker (BLC-1), Toji Field Co., Ltd.) for each cloning. .

(4) Mass culture of antibody-producing hybridoma and collection and purification of anti-rFeβ ₂ -m · mAb A large amount of the cloned hybridoma was prepared using a floating cell culture flask (filter top SC flask 250 ml 75 cm ² ; Greiner bio-one). Cultured. The culture was performed at 37 ° C., 5% CO ₂ for 5 days in a CO ₂ incubator (Toji Field Co., Ltd.), and HAT medium was used as the medium. The hybridoma cultured in large volume was suspended in serum-free RPMI and administered into the abdominal cavity of nude mice (Balb / c-nu) at 2 × 10 ⁷ cells / head. Ascites was collected 10-20 days after administration. Ascites collected from nude mice was allowed to stand at room temperature for 1 hour or at 4 ° C. overnight and then centrifuged at 3000 rpm and 4 ° C. for 5 minutes to remove fibrin, hybridoma, erythrocytes, etc. in ascites. The separated supernatant was salted out with 50% ammonium sulfate. Specifically, a saturated ammonium sulfate solution equivalent to the supernatant was gradually added dropwise with stirring on ice, and the mixture was further stirred for 1 hour. This was centrifuged at 10,000 rpm at 4 ° C. for 10 minutes, and the precipitate was dissolved in 20 mM sodium phosphate buffer (pH 7.0). The salted-out globulin solution was desalted using a Sephadex G-25 Fine (GE Healthcare Bioscience) column (inner diameter 1.5 cm, length 30 cm) equilibrated with 20 mM sodium phosphate buffer (pH 7.0). . The chromatography flow rate was adjusted to 0.5 ml / min with a peristaltic pump (SJ-1211L, ATTO). The desalted globulin solution was purified by affinity chromatography using Protein G Sepharose 4 Fast Flow (GE Healthcare Bioscience) packed in an Eco column (inner diameter 2.5 cm, length 10.0 cm: BIO-RAD). Specifically, the desalted globulin solution was added to a column equilibrated with 20 mM sodium phosphate buffer (pH 7.0) at a flow rate of 0.5 ml / min, and then the column was eluted with 100 mM glycine (pH 3.0). I let you. The eluate was immediately neutralized with 1/10 volume of 1M Tris-HCl (pH 9.0). The purified eluate was desalted with a Sephadex G-25 Fine column (inner diameter 2 cm, length 30 cm) equilibrated with 50 mM ammonium acetate (pH 7.0), and then Freeze Dryer (FDU540, EEYLA Tokyo Rika Instrument Co., Ltd.) Company) was lyophilized and stored at -20 ° C.

(5) Determination of isotype
Mouse Monoclonal Isotyping Kit (COSMO BIO INC.) Was used, and the method was performed according to the attached protocol to determine the isotype of the obtained anti-rFeβ ₂ -m · mAb. Specifically, 150 μl of the anti-rFeβ ₂ -m · mAb sample was added to the development tube, incubated at room temperature for 30 seconds, and then stirred. The isotyping strip was put therein, and further incubated at room temperature for 10 to 15 minutes, and then class and subclass were read. As the anti-rFeβ ₂ -m · mAb sample, a hybridoma culture supernatant after the second cloning was diluted 10-fold with PBS supplemented with 1% BSA. Two types of monoclonal antibodies were obtained. One antibody A isotype was IgG1 kappa chain, and the other antibody B isotype was IgG2b kappa chain.

(6) Specificity of cats to native β ₂ -m For antibodies A and B, we used a Western blotting method in which cat urine protein of chronic kidney disease (CKD) was used as an antigen. Specificity for β ₂ -m was confirmed. For comparison, a similar experiment was performed using rFeβ ₂ -m purified as described above as an antigen. Western blotting was performed in the same manner as described above. However, the urinary protein sample for electrophoresis of SDS-PAGE was a cat urine protein whose SS bond was cleaved with 2-Mercaptoethanol. FIG. 3 is a photograph showing the experimental results of the specificity of antibodies A and B against the native β ₂ -m of cat, where lane 1 shows rFeβ ₂ -m and lane 2 shows urine protein of CKD cat, respectively. . As shown in FIG. 3, both antibodies A and B were confirmed to react specifically with native β ₂ -m. CKD beta ₂ -m in urine cat, considered as than rFeβ ₂ -m purified was high molecular weight is due to beta ₂ -m in the urine of CKD cat is bound to a sugar It is done.

(7) Examination of feline nephropathy diagnosis By the sandwich method using the antibodies A and B described above, β ₂ -m in the urine of one healthy cat and three cats with chronic kidney disease was quantified. . Specifically, antibody A was solid-phased at 0.5 mg / well and blocked, and then the urine of one healthy cat and three cats with chronic kidney disease were each reacted for 2 hours. After washing, biotin-labeled antibody B was reacted for 2 hours, then avidin peroxidase was reacted for 1 hour, a substrate reaction was performed using tetramethylbenzidine, and the absorbance at 450 nm was measured. FIG. 4 is a graph showing the results, and the vertical axis represents the β ₂ -m concentration (ng / ml) in urine. As is clear from FIG. 4, the antibodies A and B of the present invention hardly reacted to the urine of healthy cats, and all three cats responded to chronic kidney disease cats. This result suggests that the urine of cats with chronic kidney disease contains a large amount of β ₂ -m, and that the antibody of the present invention can be used for diagnosis of cat nephropathy.

  The embodiments and experimental examples disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

Claims (8)

  A protein having the amino acid sequence represented by SEQ ID NO: 1.   A structural gene encoding the protein of claim 1.   A structural gene having the base sequence represented by SEQ ID NO: 2.   An antibody that specifically binds to cat-derived β2 microglobulin. The antibody according to claim 4, which is produced by the cell line Mouse-Mouse hybridoma β ₂ -mmAb1 (reception number: FERM AP-21879) using the protein described in claim 1 as an antigen. The antibody according to claim 4, which is produced by the cell line Mouse-Mouse hybridoma β ₂ -mmAb2 (reception number: FERM AP-21880) using the protein described in claim 1 as an antigen.   A kit for diagnosis of feline nephropathy, comprising the antibody according to any one of claims 4 to 6.   A method for diagnosing feline nephropathy using the antibody according to any one of claims 4 to 6.