KR100967588B1 - Protein marker related with the muscle development stage for improving economic traits of Hanwoo - Google Patents

Abstract

The present invention relates to a biomarker protein related to muscle development time and fat development time for high-quality meat production, and a method for selecting Hanwoo having excellent economic traits using the same.

According to the present invention, the biomarker protein related to the muscle development time and the fat development time of Hanwoo at the protein level can be selected using proteome analysis technology and then used to establish a new concept of advanced meat production technology. In addition, while minimizing the costly phenotypic capacity-testing operation at the same time, it can be used for the selection of high-quality meat in the early stage of growth can ensure a good Hanwoo.

Korean beef, muscle development, fat development, biomarkers, proteome, villin 2

Description

Protein marker related with the muscle development stage for improving economic traits of Hanwoo}

The present invention relates to a biomarker protein related to muscle development time, which is one of the important elements of high-quality meat selection in livestock grading, and more specifically, a protein whose expression is increased in the loin tissue during muscle development rather than the loin tissue during fat development. And it relates to a diagnostic kit that can predict the early growth trait using the same.

In general, the tissue development time of each castrated cattle starts from 2.7 months in muscle, the maximum age is 11 months, and in fat, it starts from 12.4 months and the most active period is known as 17 months. The increase is 3.84 kg and 3.85 kg fat. In particular, muscle fat content is known to increase rapidly around 17 months of age. Therefore, the discovery of protein that is specifically expressed at each time suggests that it is closely related to muscle and muscle fat development.

Recently, as the investigation of carcass traits for the production of high-quality meat has been actively conducted, interest in the intramuscular fat content in the living body is increasing. Carcass traits include carcass weight, backfat thickness, dorsal muscle cross-sectional area, carcass rate and intramuscular fat content. Therefore, intramuscular fat content can be used as an objective indicator of meat grading in livestock grading.

Therefore, the main object of the present invention is to provide a new biomarker protein for the production of high-quality beef cattle.

Another object of the present invention to provide an antibody and a diagnostic kit using the biomarker protein.

According to one aspect of the present invention, the present invention provides a selection of high-quality Korean beef, including villin 2 protein having SEQ ID NO: 1, wherein expression is increased in the lumber tissue at the time of muscle development rather than the rump tissue at the time of fat development. It provides a biomarker for.

In the present invention, "muscle development time" refers to a period between 2.7 months of age when the muscle tissue of a cow develops and 11 months of maximum developmental age, and is a time when the growth of an individual, ie, carcass weight, muscle mass, etc. increases rapidly. In addition, the "fat development time" refers to the development of muscle fat, the muscle fat content is known to increase rapidly around 17 months. Therefore, the discovery of protein that is specifically expressed at each time suggests that it is closely related to the development of muscle tissue and fat development in muscle tissue.

In the present invention, the biomarker can be used to select a Hanwoo cattle having a high intramuscular fat trait, specifically, if not detected by using a biomarker consisting of a protein that is relatively increased expression in the muscle development time The trait is a Korean cattle with high fat content, and if detected, it can be judged as a Korean cattle with low muscle fat content. That is, indirect evidence that proteins that increase or decrease specifically when muscle tissue develops when Hanwoo is growing and fat in muscle tissue starts to increase are involved in intramuscular fat synthesis and are also related to intramuscular fat mass. . Therefore, the discovered protein can be used as a biomarker that can be identified as a candidate animal having a lot of intramuscular fat accumulation and at the same time as a biomarker used for selection of high-quality meat having high intramuscular fat content.

According to another aspect of the present invention, the present invention provides a diagnostic agent for the selection of high-quality beef cattle, including an antibody that specifically binds to the biomarker protein or immunogenic fragment thereof.

In the present invention, the immunogenic fragment means a biomarker protein fragment having one or more epitopes that can be recognized by an antibody against the biomarker protein of the present invention.

In the diagnostic agent for screening high-quality beef cattle of the present invention, the antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. Polyclonal antibodies can be prepared by injecting a biomarker protein or fragment thereof that is an immunogen into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, rabbits. Immunogens are injected intramuscularly, intraperitoneally or subcutaneously, and are usually administered with an adjuvant to increase antigenicity. Blood is collected periodically from external hosts to collect serum that shows improved titers and specificity for antigens, or to separate and purify antibodies.

Monoclonal antibodies can be prepared by techniques of generation of immortalized cell lines by fusions known to those skilled in the art (Koeher and Milstein 1975, Nature, 265: 495). The manufacturing method is briefly described as follows. First, 20 μg of pure protein is immunized to Balb / C mice or a peptide is synthesized and combined with bovine serum albumin to immunize mice. The antigen-producing lymphocytes isolated from mice are then fused with myeloma in humans or mice to produce immortalized hybridomas, and hybridoma cells producing the desired monoclonal antibodies using ELISA methods. Only by propagation by selection, monoclonal antibodies are isolated and purified from the culture.

According to another aspect of the present invention, the present invention provides a method for selecting high-quality meat of Hanwoo, comprising detecting the presence of the biomarker protein in the loin tissue of Hanwoo.

In the screening method of the present invention, the detecting step directly detects the presence of the biomarker protein by 2-D electrophoresis (2-DE) from the loin tissue of Hanwoo, or makes the antigen antibody reaction by contacting the loin tissue with the antibody of the present invention. Indirectly through the presence of a biomarker protein. Currently known immunoassays (immunoassay) as an antigen antibody reaction include enzyme immunoassay (ELISA, Coated tube), magnetic particles using antibody-bound magnetic particles, latex particles using antibody-bound latex particles.

In the present invention, when judging the results using the high-quality meat selection method of the Hanwoo, the grade of the high-quality meat can be designated based on the amount of protein detected between the time of muscle development and the time of fat development based on the appropriate criteria. In other words, when the biomarker is detected based on a specific detection amount, the biomarker may be determined to be high-quality meat having an appropriate muscle thickness and intramuscular fat content if it is lower than the reference value.

According to another aspect of the present invention, the present invention provides a diagnostic kit for selection of high-quality beef cattle, comprising an antibody that specifically binds to the biomarker protein or immunogenic fragment thereof.

Diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins and the like. The antibody can be labeled by radionuclides, fluorescors, enzymes, and the like.

Monoclonal antibodies of the present invention can be used in a variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as various economic traits through the development of antibodies showing higher specificity and sensitivity It can also be used to develop protein chips with a spectrum.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Protein Extraction in Korean Beef Sirloin

The test animals used in the present invention were selected from a total of 100 castrated Hanwoo cattle, 20 individuals with the same weight, age and physiological index, and were judged at the time of muscle development (11 months old) and fat development (17 months old). The first rib area was biopsyed using a Spring-loaded biopsy instrument (Biotech Nitra, Slovak) and immediately frozen in liquid nitrogen and stored at -80 ° C until analysis. In the sample preparation process, 300 mg of sirloin tissue is added to 3 ml of lysis solution and homogenized within 1 to 2 minutes (homogenizer, IKA, Germany). The lysis solution composition was 7M Urea, 2.8M Thiourea, 4% CHAPS, 0.002% BPB, 65mM DTT, 2% IPG buffer (3-10 NL), 1X Nuclease MIX, 1X Protease inhibitor. After incubation at room temperature for 1 hour after homogenization, only the supernatant was recovered by centrifugation at 14,000 g and 20 ° C. for 30 minutes.

Example 2 Comparison of Blood Characteristics and Ultrasound in the Muscle and Fat Development Period

The blood development period (11 months of age) 10 heads and fat development (17 months old) 10 heads of blood physiological index was confirmed by growth period, the results are shown in Table 1 and 2.

TABLE 1 Blood characteristics in the development of muscle and fat

* Data are mean 10 standard ± standard deviation

TABLE 2 Ultrasound Results in Timing of Muscle and Fat Development

* Data are mean 10 standard ± standard deviation

In Table 1, the blood properties indicate that the blood physiological index of each growth period is normal. In addition, Table 2 shows that the intramuscular fat was increased through the ultrasonic waves, indicating that the intramuscular fat was increased when the ultrasonic level is increased. In other words, in the measurement of intramuscular fat using ultrasound in Table 2, 1.0 means that there is no intramuscular fat and 2.1 means that intramuscular fat began to accumulate.

Example 3 IEF (1-D Electrophoresis) and 2-D Electrophoresis

The protein extracted in Example 1 was quantified using a 2-D quant kit (GE Healthcare, USA), followed by pooling 10 heads of muscle development (11 months of age) and 10 heads of fat development (17 months of age). Dimensional electrophoresis (2-DE) was performed.

(1) First-dimension isoelectric focusing (IEF)

150 μg of the protein extracted from the tissue was subjected to rehydration with a rehydration solution (7M Urea, 2M Thiourea, 2% CHAPS, 0.002% Bromophenol blue, 65 mM DTT (Dithiotheitol), 0.5% Pharmalyte (3-10 NL; not linear)). After loading the mixture of protein and rehydration solution into a clean holder, IEF was performed using IPG strips (pH 3-10 NL and 18 cm) under the following conditions: 12 hours after rehydration Step 1: 500V, 1 hour Step 2: 1,000V, 1 hour; Step 3: 8,000V, 7 hours, Step 4: 500V, holding (21 hours in total).

(2) Second-dimension SDS-PAGE

After the completion of the IEF, each strip goes through a two-step equilibration process. The first step was reacted for 15 minutes in equilibration buffer containing 1% Dithiotheitol (DTT), and the second step was reacted for 15 minutes in equilibration buffer containing 2.5% IAA (Iodoacetamide). For reference, the equilibration buffer composition is as follows. 50mM Tris-HCl, pH8.8, 6M Urea, 30% glycerol, 2% SDS, 0.002% Bromophenol blue were used. Each strip was equilibrated on a 12.5% gel and filled with the remaining space with 0.5% agarose sealing solution. Electrophoresis conditions were completed at 16 watts after electrophoresis at 2 watts per gel until the proteins in the strip were transferred safely to the 2-d gel.

Example 4 Silver Staining and Image Scanning, Analysis

Silver staining method was used PlusOne silver staining kit (GE Healthcare, USA). For reference, the final volume per gel was 250 ml. The first step was a fixation step, in which 100 ml of ethanol, 25 ml of acetic acid and glacial were adjusted to 250 ml with tertiary distilled water, followed by about 1 hour or over night. Next, ethanol 75 ml, sodium thiosulphate 10 ml, sodium acetate (17g) 1packet was added in the sensitizing step and the final volume was 250 ml. The reaction was carried out for 30 minutes and washed three times with 10 minutes of distilled water. In the silver reaction step, add 25 ml of 2.5% (w / v) silver nitrate solution to make the final volume 250ml, add 100 μl of 37% formaldehyde just before the experiment, and react for 20 minutes, and wash 3 times with 3 distilled water for 1 minute. It was. In the developing stage, 1packet of sodium carbonate (6.25g) was added and the final volume was 250ml. Immediately before the experiment, 50 μl of 37% formaldehyde was added and reacted for 4-6 minutes. Lastly, in the stopping step, 1packet of EDTA-Na2 · 2H ₂ 0 (3.65g) was added and the final volume was 250ml for 10 minutes, followed by staining.

After scanning the stained gel for pooling fillet tissue using ImageScanner flatbed Scanner, each protein spot was identified by ProteomWeaver (DEFINIENS, Germany) and analyzed for each spot. One spot co-expressed was obtained at the time of muscle development (FIG. 1).

TABLE 3 Spot concentration analysis by ProteomWeaver

The data is a spot analysis value using the ProteomWeaver (DEFINIENS, Germany) program, the difference in the concentration of the spot expression difference in the muscle development time and fat development time shown in Table 3 can be seen in Figure 1c.

Example 5 Protein Identification Using Mass Spectrometry

Among the spots analyzed in Example 4 were selected to show a difference in protein expression level, and then trypsin treatment to in-gel digestion, using the MALDI-TOF to identify the protein, the results are shown in Table 4 and 2 is shown. The data in Table 4 are spot analyzes using the ProteomWeaver (DEFINIENS, Germany) program.

TABLE 4 Protein Identification Results by MALDI-TOF

The amino acid sequence of the identified proteins as described above is NCBInr database; was confirmed by having the villin 2 (SEQ ID NO: 1), to search is the (http // www.ncbi.nlm.nih.gov /).

Example 6 Detection of villin 2 in Hanwoo Fillet Tissues Using Immunoblotting

The pooled sirloin protein utilized in the 2DE assay of Example 2 was subjected to nitrocellulose membrane in a solution containing 25 mM Tris-HCl, 192 mM Glycine, 0.1% SDS, 20% Methanol. Transferred and transferred membrane was reacted for 1 hour at room temperature in Tri Buffered Saline Tween 20 (TBS-T) containing 5% skim milk powder. Subsequently, the mouse primary antibody IgG (Mouse monoclonal [3C12] to Ezrin, abcam) (1: 300), an Ebicam product, was reacted at 4 ° C. overnight, washed three times with TBS-T, and then produced in rabbits. Antibody IgG-HRP (Rabbit polyclonal to Mouse IgG-HRP, abcam) (1: 2,000) was reacted for 1 hour at room temperature. Finally wash three times with TBS-T. Thereafter, the nitrocellulose membrane was reacted with ECL (Enhanced chemiluminescence) to expose the X-ray film (Fig. 3a).

In Figure 3b, it was confirmed that the expression level in the sirloin tissue at the time of muscle development is more than three times higher than the loin tissue at the time of fat development, showing a similar result to the results obtained through 2DE.

As described above, according to the present invention, biomarker proteins related to muscle development time and fat development time of Korean cattle are selected at the protein level by using proteome analysis technology, and then a new concept of high-quality meat production technology can be established. have. In addition, while minimizing the costly phenotypic capacity-testing operation at the same time, it can be used for the selection of high-quality meat in the early stage of growth can ensure a good Hanwoo.

Figure 1a is a result of two-dimensional electrophoresis (2DE) analysis of the proteins expressed in the muscle development time and fat development time of the present invention.

Figure 1b is an enlarged spot with the difference in expression in the muscle development time and fat development time of Figure 1a.

Figure 2 is a result of MALDI-TOF analysis of the protein expression difference in the muscle development time and fat development time of the present invention.

Figure 3a and 3b is the result of immune blotting of the protein expression difference in the muscle development time and fat development time of the present invention.

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Claims (5)

Biomarker composition for selection of high-quality beef meat comprising a villin 2 protein having SEQ ID NO: 1, the expression is increased in the loin tissue of the muscle development time than the loin tissue of the fat development time. A diagnostic agent for screening high quality Korean beef, comprising as an active ingredient an antibody that specifically binds to the biomarker protein of claim 1 or an immunogenic fragment thereof. According to claim 2, wherein the antibody is a high-quality beef cattle diagnostic, characterized in that the monoclonal antibody. The high-quality meat selection method of Hanwoo comprising the step of detecting the presence of the biomarker protein of claim 1 in the loin tissue of Hanwoo. The diagnostic kit for selecting high-quality beef from Hanwoo, comprising an antibody that specifically binds to the biomarker protein of claim 1 or an immunogenic fragment thereof.

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