CN103376323A - Application of apolipoprotein C-III as marker of obesity-diabetes - Google Patents

Abstract

The present invention relates to the application of apolipoprotein C-III as a marker of obesity type diabetes. The present invention provides a useful marker for indicating the occurrence or development of obese diabetes—apolipoprotein C-III, whose content in the plasma of obese diabetic patients is significantly higher than that of normal people, so it can be used to develop and diagnose obesity A diagnostic reagent or kit for the risk of type 2 diabetes or obesity type diabetes.

Description

Application of Apolipoprotein C-III as a Marker of Obesity Diabetes Mellitus

technical field

The invention belongs to the field of biotechnology. Specifically, the invention relates to an application of apolipoprotein C-III (ApoC-III: Apolipoprotein C-III) as a protein molecular marker for detecting diabetes.

Background technique

Diabetes is a disease that seriously threatens human health. The incidence rate in the world is as high as 2%. In developing countries, the morbidity and mortality of diabetes are showing a rapid upward trend. In my country, with the development of social economy and the improvement of people's living standards, the prevalence of diabetes is increasing day by day. At present, the total number of people with diabetes in my country exceeds 40 million, which has become the hardest hit area for diabetes. The annual medical expenditure for diabetes treatment has greatly increased. , which shows that diabetes has become an enemy that seriously endangers the safety of people's lives and property in our country, and is an important factor affecting social and economic development. Therefore, it is of strategic significance to increase efforts to carry out basic research on diabetes in our country.

Diabetic patients can be in the pre-diagnosis of clinical diabetes for up to 12 years. Most patients are diagnosed and treated after acute or chronic complications of diabetes. In fact, early detection, early diagnosis and early treatment are the key to their recovery. Therefore, we need to find suitable markers to realize early detection of diabetes, so as to further realize early diagnosis and early treatment of diabetes; Protein molecular markers are of great significance.

With the continuous improvement of people's living standards, the number of obese people is increasing. The nationwide nutrition and health survey in 2002 showed that about 22.8% of adults over 18 years old in mainland China were overweight and 7.1% were obese; increased by 40.7% and 97.2%, respectively (Chen 2008). Studies have shown that obesity may directly cause many diseases, or greatly increase the risk of certain diseases. And, as body mass index (BMI) rises, the risk gradually increases. Increased body mass index is an important risk factor for chronic diseases such as cardiovascular system disease, type 2 diabetes, and respiratory system disease (Must, Spadano et al. 1999; Kopelman 2000; Kushner and Roth 2003; Kushner and Blatner 2005). Among chronically obese people, the prevalence of diabetes is more than five times that of the general population.

Therefore, it is very necessary in this field to find markers for early diagnosis of obesity-related diabetes or for predicting the risk of obesity-related diabetes in various ways, in order to provide a basis for clinical diagnosis.

Contents of the invention

The object of the present invention is to provide the application of apolipoprotein C-III (Apo C-III: Apolipoprotein C-III) as a protein molecular marker for detecting obesity-type diabetes.

In the first aspect of the present invention, a use of apolipoprotein C-III as a marker of obesity-type diabetes is provided.

In another preferred example, the apolipoprotein C-III is used as a protein molecular marker for detecting obesity diabetes or predicting the occurrence of obesity diabetes.

In another preferred example, the markers are markers of body fluids.

In another preferred example, the body fluid is plasma or serum.

In another preferred example, the apolipoprotein C-III also includes its protein fragment, and the amino acid sequence of the protein fragment is unique to apolipoprotein C-III.

In another preferred example, the amino acid sequence of the protein fragment of apolipoprotein C-III is shown in SEQ ID NO: 4 or amino acids 2-12 thereof.

In another aspect of the present invention, an isolated polypeptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 4 or amino acids 2-12 thereof.

In another aspect of the present invention, an isolated polynucleotide encoding said polypeptide is provided.

In another aspect of the present invention, a use of apolipoprotein C-III for preparing a reagent for detecting obesity-type diabetes is provided.

In another preferred example, the reagent is selected from: antibodies and ligands.

In another preferred example, the antibodies include monoclonal antibodies and polyclonal antibodies.

In another aspect of the present invention, a use of a reagent for specifically recognizing apolipoprotein C-III or its protein fragments is provided for preparing a kit for detecting obese diabetes or distinguishing high-risk groups of obese diabetes.

In another preferred example, the reagent specifically recognizing apolipoprotein C-III or its protein fragment is an antibody.

In another aspect of the present invention, a reagent for specifically recognizing apolipoprotein C-III or its protein fragment is provided, and the reagent is a polyclonal antibody.

In another aspect of the present invention, a kit for detecting obesity-related diabetes is provided, the kit comprising a container, and the reagent placed in the container.

In another aspect of the present invention, a test strip (such as a test paper or a test strip) is provided, which includes a solid-phase carrier, and the reagent attached to the solid-phase carrier.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. The MS/MS spectrum of the peptide GWVTDGFSSLD used to detect the concentration of apolipoprotein C-III in plasma in the mass spectrometry multiple reaction monitoring technique.

Figure 2, 598.8_854.4 (y8) the distribution of R ² of the parent ion-product ion pair in 145 samples. The abscissa indicates the ^R2 value, and the ordinate indicates the number of occurrences of different ^R2 values.

Fig. 3, the result of mass spectrometry multiple reaction monitoring technique to apolipoprotein C-III parent ion-product ion pair (598.8_854.4). The ordinate is the ratio of the light peptide divided by the heavy peptide (L/H), that is, the ratio of the blood content of the peptide GWVTDGFSSLD to the standard peptide. Two groups were tested by t-test, p value<0.0001, ** means p<0.01.

Figure 4. ROC curve of MRM results of apolipoprotein C-III.

Detailed ways

After extensive research and screening, the inventor finally obtained a useful marker for indicating the occurrence or development of obesity-type diabetes. The marker is apolipoprotein C-III (Apolipoprotein C-III, Apo C-III) or its protein fragment, and its content in the body fluid of obese diabetic patients is significantly higher than that of normal people, so it can be used to diagnose obese Diabetes or obesity-related diabetes risk.

apolipoprotein C-III

The Genebank accession number of apolipoprotein C-III is GI: 4557323, the NCBI accession number is: NP_000031.1, the Swissprot accession number is: P02656, and the IPI number is: IPI00021857. Apo C-III is a glycoprotein composed of 79 amino acids, mainly synthesized in the liver, followed by the small intestine, and is an important component of high-density lipoprotein and triglyceride-rich lipoprotein. Apo C-III is known to inhibit the activity of lipoprotein lipase and hepatic lipase, thereby delaying the clearance of triacylglycerol-rich lipoproteins. Apo C-III can competitively inhibit the binding of Apo E to the liver Apo E receptor, and hinder the Apo E-mediated uptake of chylomicron CM and very low-density lipoprotein VLDL by the liver. Therefore, the abnormal expression or secretion of Apo C-III will cause abnormal lipid metabolism. There is currently no report on the use of apolipoprotein Apo C-III as a molecular marker for human obesity-related diabetes.

Based on the new discovery of the present invention, a peptide segment (polypeptide) derived from apolipoprotein C-III is provided. The peptide segment is unique to the apolipoprotein C-III protein and can be well applied as a molecular markers. Preferably, the peptide segment has the amino acid sequence shown in SEQ ID NO: 4, or has the amino acid sequence shown in positions 2-12 of SEQ ID NO: 4. The present inventor analyzed a large number of peptides derived from apolipoprotein C-III, and it was identified that this peptide has the best intensity. Detecting the content of this peptide can directly reflect the content of apolipoprotein C-III protein, which is convenient, fast and highly accurate.

The polynucleotide encoding the peptide of apolipoprotein C-III is also included in the present invention. Since the peptide is unique to the apolipoprotein C-III protein, its corresponding polynucleotide sequence is also unique. The content of the polynucleotide in the sample to be tested can be known by conventional nucleic acid detection methods.

Methods for detecting the content of polypeptides or nucleic acids are well known to those skilled in the art. For example, the detection of polypeptides can be by means of mass spectrometry instruments, or by methods such as Western Blot or ELISA. Nucleic acid detection includes methods such as PCR amplification and Northern Blot.

Uses of apolipoprotein C-III or protein fragments thereof

The inventors analyzed the plasma samples of normal healthy control group and obese diabetic patients (both obtained from the Institute of Nutrition, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) by ethanol precipitation fractionation-cation exchange chromatography-reverse phase chromatography tandem mass spectrometry analysis strategy. The protein was identified, and apolipoprotein C-III was successfully identified in both normal human plasma and obese diabetic human plasma, which provided possible sequence information for subsequent mass spectrometry multiple reaction monitoring technology MRM experiments. Through mass spectrometry multiple reaction monitoring technology, it was further confirmed and found that the expression levels of apolipoprotein C-III in the plasma of the normal healthy control group and the obese diabetic group were differentially expressed.

By detecting the expression level of apolipoprotein C-III in the plasma of normal healthy controls and obese diabetic patients, the inventors found that there was a differential expression of apolipoprotein C-III in the plasma of normal healthy controls and obese diabetic patients. The expression level of C-III in the plasma of obese diabetic patients is significantly higher than that in the plasma of normal healthy controls; through MS/MS detection, database search, protein search, buildsummary analysis, MRM detection, and quantification of apolipoprotein C-III , It was further confirmed and found that the expression levels of apolipoprotein C-III in the normal healthy control group and the obese diabetic group were differentially expressed.

Therefore, the first object of the present invention is to provide an application of apolipoprotein C-III or its protein fragment as a protein molecular marker for detecting or predicting the occurrence of obese diabetes.

Therefore, the present invention provides a use of apolipoprotein C-III or its protein fragment as a marker of obesity-type diabetes. Those skilled in the art understand that apolipoprotein C-III is a multifunctional protein distributed on the surface of blood, ascites fluid, cerebrospinal fluid, and various types of cells. Apolipoprotein C-III can be used as a molecular marker. As a preferred mode of the present invention, apolipoprotein C-III is used as a plasma detection marker for obesity-type diabetes. Plasma testing is convenient, easy to operate, easy to review, easy to accept by patients, evaluates prognosis, and guides treatment.

Based on the new discovery of the present invention, apolipoprotein C-III protein can be used to: (i) carry out differential diagnosis and/or susceptibility analysis of obesity diabetes; , prognosis, and selection of appropriate treatment methods; (iii) early assessment of the risk of obesity-related diabetes in relevant populations, early monitoring and early prevention.

It can also be used to prepare reagents that specifically recognize apolipoprotein C-III, so as to detect the existence and amount of apolipoprotein C-III as the basis for judging obesity-type diabetes.

Reagents and kits for recognizing apolipoprotein C-III

Based on the new discovery of the present invention, the present invention also provides a reagent for specifically recognizing apolipoprotein C-III or its protein fragment. Any reagent that can recognize apolipoprotein C-III or its protein fragments is included in the present invention and used as a marker for detecting obesity-type diabetes. The reagent that specifically recognizes apolipoprotein C-III or its protein fragments is, for example, an antibody or ligand that specifically binds apolipoprotein C-III.

The present invention also provides the use of a reagent for specifically recognizing apolipoprotein C-III or its protein fragments for detecting obese diabetes or obese diabetic high-risk groups.

As an embodiment of the present invention, the reagent is an antibody against apolipoprotein C-III; more specifically, it is a polyclonal antibody.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified antigen can be administered to animals such as rabbits, mice, rats, etc. to induce polyclonal antibody production. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).

The antibody can be used in immunohistochemical technique to detect the level of apolipoprotein C-III in the sample, so as to diagnose obese diabetes or judge the risk (susceptibility) of the disease.

The antibody can be used to detect the level of apolipoprotein C-III in body fluids, and thus can be used to detect obesity-related diabetes, can be used to predict the occurrence of early-stage obesity-related diabetes, or can be used to prepare preparations or kits for detecting obesity-related diabetes etc., for preparing preparations or kits for predicting the occurrence of early obesity-type diabetes.

The present invention also provides a kit for diagnosing obesity-type diabetes, which includes: a reagent for specifically recognizing apolipoprotein C-III or its protein fragment. The reagents are, for example, monoclonal antibodies or polyclonal antibodies.

The kit may also contain: reagents for immunohistochemical analysis, such as: secondary antibodies, staining reagents, chromogenic reagents and the like. In addition, the kit may also include instructions for use and the like.

More specifically, the kit may be a kit based on enzyme-linked immunoassay (ELISA) technology. It is used to detect obesity-related diabetes or predict the occurrence of early-stage obesity-related diabetes. ELISA techniques and detection reagents based on this technique will be apparent to those skilled in the art.

More specifically, the reagent that specifically recognizes apolipoprotein C-III or its protein fragments can also be immobilized on a test paper to prepare immunocolloidal gold test paper or similar detection materials.

The main advantages of the present invention are:

(1) It is revealed for the first time that apolipoprotein C-III or its protein fragments can be used as a marker for the diagnosis of obesity-related diabetes.

(2) For the first time, a reagent for specifically recognizing apolipoprotein C-III or its protein fragments has been prepared. The reagent has good specificity, high detection sensitivity, high accuracy, and has good clinical application value.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, generally according to conventional conditions such as people such as Sambrook, molecular cloning: the condition described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 2002), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1. Screening method for differentially expressed proteins-ethanol precipitation method fractionation-cation exchange chromatography-reverse phase chromatography tandem mass spectrometry strategy

All experimental water in this embodiment all comes from Milli-Q (Millipore, Bedford, MA, USA) ultrapure water device; dithiothreitol (DTT), SDS, urea, guanidine hydrochloride, 3-[(3-cholamidopropyl) -Diethyl]-propanesulfo (CHAPS), Tris, ammonium bicarbonate (NH ₄ HCO ₃ ) and iodoacetamide (IAA) were purchased from Bio-Rad (Herc μLes, CA, USA); Trypsin was purchased from Promega Company (Madison, WI, USA); formic acid (FA), trifluoroacetic acid (TFA) and acetonitrile (Acetonitrile) were purchased from Aldrich Company (MilWaukee, WI, USA); phosphoric acid was purchased from Shanghai Chemical Reagent Company . Except for acetonitrile, which was chromatographically pure, the rest of the chemical reagents were analytically pure.

Add 750 μL solution (100 mM NaCl, 10 mM Tris, pH 7.4) to 150 μL plasma sample, and use a 0.22 μm filter membrane to remove lipids by centrifugation at 4°C and 10,000 g; take 780 μL fat-free plasma sample and add 565 μL cold ethanol solution , placed on a vertical shaker at 4°C for 1 h, and then centrifuged at 16,000 g for 45 min at 4°C. The supernatant is the high-abundance component, and the precipitated part is the low-abundance component. Rinse the low-abundance components with 100% (v/v) cold ethanol once, freeze-dry at low temperature, add 600 μL 6M guanidine hydrochloride solution to dissolve, take 300 μL, add 5 μL 1M DTT, mix well, place at 37°C for 2.5 hours, and cool After reaching room temperature, 25 μL of 1M IAA was added, and placed at room temperature in the dark for 40 minutes. After the above treatment, the protein is completely denatured, the disulfide bonds are opened, and the sulfhydryl groups are blocked. Then add 1.6ml of cold protein precipitation solution (acetone:ethanol:acetic acid=50:50:0.1), place at -20°C for 15h, centrifuge at 15,000g at 4°C for 45min, remove the supernatant, add 1mL of 100% (v/v) cold acetone , shake for 1 min, centrifuge at 15,000 g at 4°C for 45 min, remove the supernatant, add 1 mL of 70% cold ethanol, shake for 1 min, centrifuge at 15,000 g at 4°C for 45 min, remove the supernatant, freeze-dry, add 250 μL of 100 mM ammonium bicarbonate solution, 50 μg of trypsin, 37°C After 4 hours of enzymatic hydrolysis, 50ug of trypsin was added again, and the enzyme was hydrolyzed at 37°C for 16 hours. Transfer the liquid in the EP tube to a Millipore 10K ultrafiltration tube, ultrafilter at 10,000 g at 4°C for 45 minutes, remove enzymes and unenzymolyzed proteins, collect the filtrate, freeze-dry, redissolve each tube with 0.1% formic acid aqueous solution, and take the original plasma protein enzyme The hydrolysis product (equal mass) 50ug loads mass spectrometry detection.

Cation-exchange chromatography-reversed-phase chromatography tandem mass spectrometry (SCX-RP-MS/MS) consists of an autosampler, two high-pressure mixing pumps, and a cation-exchange chromatography separation column (SCX column) for two-dimensional liquid chromatography separation and C18 reversed-phase chromatographic separation column (RP column) and two C18 reversed-phase trap columns, a ten-way valve. High-performance liquid chromatography solution includes two kinds: 1. Cation exchange chromatography separation solution, including A: pH 2.5 solution; B: pH 8.5 solution; 2. Reverse phase chromatography separation solution: A: 0.1% (v/v) formic acid; B : 0.1% formic acid, 100% acetonitrile. The peptide mixture was first washed with eluting solutions with different pH values from low to high, and the peptides eluted under different pH gradients were separated by reversed-phase chromatography, and then detected by mass spectrometry. The specific experimental process is as follows. The peptide mixture after enzymatic hydrolysis enters the SCX column in a lossless mode through the autosampler. Under the washing of the eluent at a certain pH value, the unretained peptides are washed to the No. 1 C18trap column behind After 180 minutes, the ten-way valve is switched, and the next pH gradient is eluted on the SCX column, and the eluted peptides are bound to the No. 2 C18trap column. At the same time, wash with 2-35% 100% acetonitrile solution for one No. C18 trap column, and the peptides eluted on the trap were separated again on the C18 reversed-phase chromatographic separation column, and detected by mass spectrometry. After 180 minutes, the ten-way valve was cut again. At this time, the peptides eluted by the pH gradient The peptides are bound to the No. 1 C18trap column, and the peptides eluted from the previous pH gradient eluted on the No. 2 C18trap column are washed with 2-35% 100% acetonitrile solution, and the peptides eluted from the trap are The segment is separated again on a C18 reverse-phase chromatographic separation column, and detected by mass spectrometry, and so on. A total of 10 different pH gradients were used to elute the peptides on the SCX column. The mass spectrometry conditions in the whole process are: the mass spectrometry scanning conditions are set to a full scan (full scan) of 400-2000M/z followed by MS/MS scanning of the first 10 highest peaks in the full scan, wherein dynamic exclusion (dynamic exclusion) The settings are: the repeat count is 2, the repeat duration is 30s, and the dynamic exclusion duration is 120s. Mass spectrometry was repeated once.

Thirty-three plasma samples were used in the experiment, including: 5 samples of normal weight and normal blood sugar (group 1), 5 samples of normal weight and hyperglycemia (group 2), 5 samples of normal weight and diabetes (group 3), 6 samples of Obese and normoglycemic samples (group 4), 6 obese and hyperglycemic samples (group 5), 6 obese diabetic samples (group 6). For each group of samples, an equal volume of plasma sample was taken out and mixed into a 150 μL plasma mixed sample, a total of six samples were obtained, and the above-mentioned experimental operations were carried out respectively. The clinical indicators of all samples are listed in Table 1. The clinical indicators measured were: fasting blood glucose (FPG, mmol/l), insulin (Insulin, μU/ml), triglyceride (TG, mmol/l), high-density lipoprotein cholesterol (HDL_C, mmol/l), Low-density lipoprotein cholesterol (LDL_C, mmol/l), total cholesterol (TC, mmol/l),

Table 1. Clinical data of 33 plasma samples

Embodiment 2 , mass spectrometry result analysis

Use IPI human 3.51 to search the library for the original data obtained by the two mass spectrometry detections in the above example, and make a buildsummary. The peptide screening strategy is the parameters of peptide FDR<=1% and unipeptides>=2. A total of 717 proteins were identified. 31 were identified in the library with a protein FDR of 4.32%.

Table 2 shows the number of identifications of peptides of apolipoprotein C-III (specific to apolipoprotein C-III) in the 6 groups of plasma. The peptides in the table are all enzymatically degraded apolipoprotein C-III ( The peptide sequence obtained from protein C-III).

Figure 1 shows the MS/MS spectrum of the unique peptide GWVTDGFSSLOK used to detect apolipoprotein C-III in plasma in the mass spectrometry multiple reaction monitoring technique. It has been identified that this peptide has a high intensity, a high number of detected peptides, and a moderate number of amino acids, which is suitable for the detection and verification of mass spectrometry multiple reaction monitoring technology (usually mass spectrometry multiple reaction monitoring technology can detect amino acids containing 7-22 peptides).

Table 2, Number of peptides identified by mass spectrometry of apolipoprotein C-III in plasma of 6 groups

In Table 2, "." indicates the cleavage site of trypsin; trypsin specifically recognizes and cleaves the peptide bond formed by carboxyl groups of K and R. After enzyme digestion, the peptide segment between two "." is obtained.

The above table shows that the number of peptide identifications of apolipoprotein C-III in blood plasma of obese diabetic patients is 26 times, which is higher than that of normal people's plasma of 23 times. Through this example, apolipoprotein C-III was successfully identified in normal human plasma and obese diabetic human plasma, which provided necessary sequence information for subsequent MRM experiments. Later use MRM to verify.

Example 3. Validation of mass spectrometry multiple reaction monitoring technology for differential expression of apolipoprotein C-III

Dithiothreitol (DTT) used in this example was purchased from Sigma; iodoacetamide (IAA), formic acid were purchased from Fluka; acetonitrile was purchased from Merck; capillary reversed-phase liquid chromatography, triple quadrupole mass spectrometry All were purchased from Agilent Company. The standard heavy-labeled peptide GWVTDGFSSLDK*(Heavy-Lys) was synthesized by conventional methods ("*" indicates heavy-labeled amino acids).

Add 97 μL 2D lysate (8M urea, 40mM Tris, 65mM DTT) to every 3 μL stock solution of the plasma sample, place at 4°C for 1 hour, add 1 μL 1M DTT, and place at 37°C for 2.5 hours, after cooling to room temperature, add 15 μL 1M IAA, Place at room temperature in the dark for 40 minutes, transfer the sample to a Millipore10K ultrafiltration tube, centrifuge at 10,000 g at 4°C for 45 minutes, add 100 μL of 50 mM ammonium bicarbonate solution, centrifuge at 10,000 g at 4°C for 45 minutes, then add 12 μg of trypsin, and 76 μL of 50 mM ammonium bicarbonate solution, Enzymolysis at 37°C for 16 hours, then transfer the membrane to another centrifuge tube, centrifuge at 10,000g at 4°C for 45min, add 100μL of 50mM ammonium bicarbonate solution, centrifuge at 10,000g at 4°C for 45min, collect the centrifuged liquid twice, add 3μL formic acid , freeze-dried. The freeze-dried peptide samples were dissolved with 100 μL of 0.1% (v/v) FA solution.

Take the plasma protein hydrolyzate (equal volume) corresponding to 0.3 μL of the original plasma and 20 pmol of the standard heavy-labeled peptide, mix them and add them to the reversed-phase column in a lossless mode through an autosampler. The solvent used for the reversed-phase column is 0.1% (v/v) formic acid aqueous solution (solution A), 0.1% (v/v) formic acid, 90% (v/v) acetonitrile solution (solution B). At a flow rate of 1.5 μL/min, the reverse-phase column was loaded with 5% solution B in 0-20 min, and gradient eluted with 5-35% (volume ratio) solution B in 20-120 min. The sample eluted from the reversed-phase column was detected by a nanospray ion source into QQQ 6410 (Agilent). Select 598.8_147.1(y1), 598.8_434.3(y4), 598.8_638.4(y6), 598.8_753.4(y7), 598.8_854.4(y8), 598.8_953.5(y9) in total 6 A parent ion-product ion pair was detected, the signal at the co-elution position was taken as a positive signal and its peak area was integrated, and finally the quantitative information of the parent ion-product ion pair 598.8_854.4(y8) was taken as the peptide segment Quantitative information for the follow-up data analysis, the correlation between the light peptide and heavy peptide signal intensities of the parent ion-product ion pair 598.8_854.4(y8) within the elution time in all samples is shown in Figure 2. Table 3 shows 36 cases of normal healthy control plasma apolipoprotein C-III (Apolipoprotein C-III) MRM mass spectrometry validation data, table 4 shows 109 cases of obesity diabetic plasma apolipoprotein C-III (Apolipoprotein C-III ) MRM mass spectrometry verification data, the ratio in the table is the ratio of light peptide divided by heavy peptide, that is, the value of the peptide content in blood compared to the standard peptide, ^R2 is the correlation between the signal intensity of light peptide and heavy peptide, where 100% of the data R ² >= 0.85.

table 3

Table 4

The quantitative information of the parent ion-product ion pair (598.8_854.4) was taken as the quantitative information of the peptide (GWVTDGFSSLD) for subsequent data analysis. The parent ion-product ion pair (598.8_854.4) obtained ratio of normal people and obese diabetics is done t-test test, p value＜0.0001, less than 0.01, has verified that apolipoprotein C-III is in obese diabetics. The protein content in plasma was significantly higher than that of normal people, as shown in Figure 3.

Use the MRM results to make the ROC curve, the results are shown in Figure 4, according to the figure, the area under the curve: 0.8262, apolipoprotein C-III cut point = 1.309, sensitivity = 0.7064, false positive rate = 0.1944, specificity = 0.8056.

To sum up, apolipoprotein C-III is significantly increased in the plasma of obese diabetic patients, which is obviously closely related to the occurrence and development of obese diabetes. Therefore, the expression of this protein as a molecular marker can be used to detect obesity Prediction of onset and development of type 2 diabetes.

Embodiment 4 , the preparation of antibody

The inventor injected the full-length apolipoprotein C-III protein (GenBank accession number GI: 4557323) into rabbits to obtain immune serum. The specific preparation method is as follows:

Apolipoprotein C-III protein and Freund's adjuvant were mixed at a volume ratio of 1:1, and then injected into rabbits with an immunization dose of 0.5 mg, immunized once every 2 weeks, and immunized three times. The rabbit polyclonal antibody against apolipoprotein C-III protein was obtained after routine affinity purification.

Western blot experiments show that the obtained rabbit polyclonal antibody only recognizes the apolipoprotein C-III protein, basically does not recognize other proteins, and can be used as a detection reagent for detecting obesity-related diabetes or the risk of obesity-related diabetes.

The obtained polyclonal antibody, secondary antibody (conventional), and chromogenic reagent are respectively placed in different containers, and the containers and instructions for use are placed in a box to obtain a detection kit.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. an apoC-III is as the purposes of obese diabetic mark.

2. purposes as claimed in claim 1 is characterized in that, described apoC-III also comprises its protein fragments, and the amino acid sequence of this protein fragments is peculiar by apoC-III.

3. purposes as claimed in claim 2 is characterized in that, the amino acid sequence of the protein fragments of described apoC-III is such as SEQ ID NO:4 or wherein shown in the 2-12 amino acids.

4. the polypeptide of a separation is characterized in that, its amino acid sequence is such as SEQ ID NO:4 or wherein shown in the 2-12 amino acids.

5. the polynucleotide of a separation is characterized in that, its polypeptide claimed in claim 4 of encoding.

6. the purposes of an apoC-III is for the preparation of the reagent that detects obese diabetic.

7. the purposes of the reagent of a specific recognition apoC-III or its protein fragments is for the preparation of detecting obese diabetic or distinguishing obese diabetic people at highest risk's kit.

8. the reagent of a specific recognition apoC-III or its protein fragments, described reagent is polyclonal antibody.

9. kit that detects obese diabetic, described kit comprises container, and the reagent claimed in claim 8 that places described container.

10. test-strips, it comprises solid phase carrier, and is attached to the reagent claimed in claim 8 on the described solid phase carrier.

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