Cardiac troponin i fragment, product prepared using same, and use thereof

Abstract

Provided are a cardiac troponin I fragment, a product prepared using same, and the use thereof. The specific technical solution comprises: a cTnI (31-164) protein fragment, an HSP65 (187-375) protein fragment, and a protein fragment combination. Provided is a protein antigen or composition which can be used for detecting acute myocardial infarction, has ideal detection specificity and sensitivity, and can be further prepared into a related reagent or kit on the basis of need.

Description

Cardiac troponin I fragments, products prepared therefrom and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, specifically cardiac troponin I fragments, products prepared therefrom and uses thereof. BACKGROUND

[0002] Acute myocardial infarction (AMI) is the most urgent acute coronary syndrome. It is mainly caused by the rupture of the coronary atherosclerotic plaque, which leads to the exposure of a large amount of thrombogenic material, and then the acute occlusion of the coronary lumen by aggregated platelets, fibrin and red blood cells. The incidence and mortality of acute myocardial infarction are high.

[0003] At present, biochemical detection has become one of the important means for detecting acute myocardial infarction in clinical practice. The developed biomarkers include serum glutamate oxaloacetate transaminase / aspartate transaminase (SGOT / AST), lactate dehydrogenase (LDH), myoglobin, creatine kinase (CK), creatine kinase MB isozyme (CK-MB) and troponin (Tropoin) and the like. However, the sensitivity and specificity of these markers in detection need to be improved, and the available biomarkers are limited.

[0004] Therefore, if the available range of biomarkers can be expanded and the sensitivity and specificity of the detection of acute myocardial infarction can be improved, it will have great application prospects. SUMMARY

[0005] The purpose of the present application is to provide cardiac troponin I fragments, products prepared therefrom and uses thereof.

[0006] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is: a cTnI (31-164) protein fragment, wherein the amino acid sequence of the cTnI (31-164) protein fragment is shown as SEQ ID NO: 1.

[0007] Correspondingly, an antigen combination includes a HSP65 (187-375) protein fragment, a TNNC1 protein fragment, a DCD (20-110) protein fragment and a NPY (29-97) protein fragment.

[0008] The amino acid sequence of the HSP65(187-375) protein fragment is shown as SEQ ID NO: 5, the amino acid sequence of the TNNC1 whole protein is shown as SEQ ID NO: 3, the amino acid sequence of the DCD(20-110) protein fragment is shown as SEQ ID NO: 6, and the amino acid sequence of the NPY(29-97) protein fragment is shown as SEQ ID NO: 8.

[0009] Correspondingly, the HSP65(187-375) protein fragment has an amino acid sequence shown as SEQ ID NO: 5.

[0010] Correspondingly, the cTnI(31-164) protein fragment or the antigen combination or the HSP65(187-375) protein fragment is used for preparing a product for detecting / identifying acute myocardial infarction and / or drug-induced myocardial injury.

[0011] Correspondingly, a reagent or kit for detecting / identifying acute myocardial infarction and / or drug-induced myocardial injury is prepared by using the cTnI(31-164) protein fragment or the antigen combination or the HSP65(187-375) protein fragment.

[0012] Correspondingly, a reagent or kit for detecting / identifying acute myocardial infarction and / or drug-induced myocardial injury is prepared by using the cTnI(31-164) protein fragment or the antigen combination or the HSP65(187-375) protein fragment.

[0013] In the drug-induced myocardial injury, the drug is a drug for treating tumors or cancer.

[0014] The present application has the following beneficial effects: the present application provides an antigen for detecting acute myocardial infarction / myocardial injury related autoantibodies: cTnI (31-164), HSP65 (187-375), which can be used to detect whether there is an autoantibody against the above antigen combination in a biological sample from a subject, so as to determine whether the subject has acute myocardial infarction / myocardial injury or has a potential risk of developing acute myocardial infarction / myocardial injury. Compared with cTnI full protein and other protein fragments of cTnI, the sensitivity of cTnI (31-164) is significantly improved while maintaining high specificity, and the clinical application value is also significantly improved. The sensitivity of HSP65 (187-375) is slightly lower than that of HSP65 full protein and HSP65 (1-540), but the specificity is obviously improved, which can reduce the possibility of false positives while ensuring the detection rate. Therefore, cTnI (31-164), HSP65 (187-375) can be used alone or in combination with other biomarkers for detecting / diagnosing acute myocardial infarction, which can be used for detecting myocardial infarction and chronic myocardial injury (such as myocardial loss caused by antitumor drugs), and can also be used for assisting in identifying coronary heart disease and guiding the use of clinical drugs. Among them, the biological sample can be serum, plasma, whole blood, saliva, oral mucosa swab, urine, lymph, cerebrospinal fluid, etc. According to the specific circumstances, the biological sample can be pretreated by extraction, dilution, enrichment, etc. The method is various and easy to operate. DETAILED DESCRIPTION

[0015] The present application first found that the cTnI (31-164) protein fragment is closely related to acute myocardial infarction and myocardial injury, and the cTnI (31-164) protein fragment is used for detecting acute myocardial infarction, which has high specificity and ideal sensitivity, and can be used alone or in combination with other biomarkers for preparing detection and diagnosis products (such as kits) related to acute myocardial infarction.

[0016] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. If not specifically indicated, the technical means used in the embodiments are conventional means familiar to those skilled in the art. The obtained data are all average values obtained after at least 3 repetitions, and each repetition obtains valid data.

[0017] Example 1: Recombinant vector construction, expression and purification of antigen The method for obtaining antigen can be selected as follows: synthesizing the DNA encoding protein antigen, taking the synthetic DNA as template, designing primers, cloning the gene fragment of the protein antigen or its fragment to the expression plasmid through PCR, enzyme digestion, ligation and other molecular cloning means, then expressing through E. coli, yeast or cells, and then purifying the target protein through chromatography. Meanwhile, the Trx, GST, AVI, HIS, c-myc and other tags can be selectively added to the protein antigen or its fragment. Adding these tags can facilitate the purification or labeling of the protein antigen, but will not change the binding properties of the antigen and its own antibody in essence.

[0018] The potential proteins related to the detection of acute myocardial infarction were selected, and the database IDs of the respective proteins are shown in Table 1.

[0019] Table 1: Database ID of each potential protein

[0020] Taking the human cDNA library (purchased from Invitrogen Corporation) or the whole gene synthetic DNA as template, primers were designed respectively, and the gene fragment of the protein was cloned to the pET28 plasmid through PCR, enzyme digestion, ligation and other molecular cloning means. Meanwhile, the HIS, FLAG and other tags were added to the N-terminus of the protein to form a fusion protein. The obtained recombinant expression vector was identified by DNA sequencing to confirm that it contained the correct protein gene fragment. It should be noted that adding the tag is only for the convenience of identifying and extracting the protein, and does not have a decisive influence on the function of the protein as an antigen. When used, no tag is added or other tags are added or other labeling / identification means are adopted.

[0021] The recombinant plasmid containing the protein gene fragment was transformed into E. coli BL21 (DE3) competent cells, and the clones were inoculated into LB medium and cultured at 37°C on a shaker. When the bacterial density OD 600 was about 0.8, the temperature was lowered to 16°C, 0.1 mM isopropyl thiogalactoside (IPTG) was added to each LB medium, and the expression was induced overnight to obtain the bacterial cells.

[0022] The bacteria bodies of the induced expression were collected by centrifugation and rinsed twice with PBS. The bacteria bodies were resuspended and dispersed with lysis solution (5-10 mL of lysis solution per g of bacteria bodies), ice-bathed, and ultrasonically broken (ultrasonic power 200 W, breaking for 5 s and resting for 5 s). After breaking, the bacteria bodies were centrifuged at 13000 rpm and 10°C for 20 min, and the supernatant was taken. After two-step purification of the supernatant by Ni column affinity chromatography and molecular sieve chromatography, the molecular weight and purity of the protein were confirmed by SDS-PAGE electrophoresis analysis, the concentration was determined by the Bradford method, and the protein was stored at -80°C for standby. Thus, the purified candidate proteins were obtained. Among them, the whole proteins of cTnI, TNNC1 and HSP65 were obtained; the protein fragments of HBEGF (19-160), NPPA (26-151), DCD (20-110) and NPY (29-97) were obtained; and the whole protein of VEGFR1 was not obtained.

[0023] Example 2: Effect of each candidate protein on detection of acute myocardial infarction samples

[0024] (2) Blocking solution / sample diluent / antibody diluent: 10 g of BSA (bovine serum albumin) was dissolved in 800 ml of coating buffer, and the coating buffer was added to 1 L.

[0025] (3) Washing solution: prepared immediately before use, 0.5% Tween 20 (V / V) was added to the coating buffer before use, pH = 7.4.

[0026] (4) TMB color developing agent, purchased from KPL company.

[0027] (5) Stop solution: 1M hydrochloric acid.

[0028] 2. Solid-phase coating of the protein to be tested. The purified candidate proteins obtained in Example 1 were diluted to 5 μg / mL with coating buffer, added to a 96-well plate at 50 μL per well, and coated at 4°C overnight. The next day, the solution was poured out, spun dry, and washed three times with washing solution at 200 μL per well each time. Then 200 μL of blocking solution was added to each well, incubated at room temperature for 1 h, the blocking solution was poured out, spun dry, and washed three times with washing solution at 200 μL per well each time and spun dry again; thus, the solid-phase coated antigen in the 96-well plate was obtained.

[0029] 3. Add the sample to be tested. Dilute the human serum to be tested 100 times with the sample dilution solution, and add 50 μL of the diluted sample to be tested to each well of the 96-well plate containing the protein to be tested. Then, place the 96-well plate on a microplate shaker, and incubate at room temperature for 1 hour with shaking. After shaking, wash the plate three times with 200 μL of the washing solution per well, and shake again.

[0030] 4. Add the enzyme-labeled secondary antibody. Dilute 1.0 mg / mL of horseradish peroxidase-labeled recombinant goat anti-human immunoglobulin G antibody (purchased from Jackson ImmunoResearch Inc.) 20,000 times with the antibody dilution solution, and add 50 μL of the diluted antibody to each well of the 96-well plate treated in step 3. Then, place the 96-well plate on a microplate shaker, and incubate at room temperature for 0.5 hour with shaking. After shaking, shake the plate again, wash the plate three times with 200 μL of the washing solution per well, and shake again.

[0031] 5. Color development and reading of optical density values. Add 50 μL of TMB color developing reagent to each well of the 96-well plate treated in step 4, shake for 15 seconds, and allow to react at room temperature for 15 minutes in the dark. Then, add 50 μL of a stop solution, and read the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader to obtain the detection signal (S) of each sample to be tested.

[0032] 6. Sensitivity and specificity analysis. Take 384 positive samples (serum samples of patients who have been diagnosed as having myocardial infarction) and 384 negative samples (serum samples of healthy subjects), and measure the detection signal (S) of each sample according to the above method (absorbance at 450 nm). Calculate the average (M) and the standard deviation (SD) of the detection signals (S) of all negative reference samples using the negative samples as the negative reference samples, and set M+3SD as the Cut Off value. Samples with a detection signal (S) of ≥Cut Off value (S≥M+3SD) are defined as positive, and samples with a detection signal (S) of <Cut Off value (S

[0033] Calculate the specificity and the sensitivity based on the positive and negative results of the samples. The specificity is the proportion of samples of healthy subjects that are correctly determined to be negative, i.e., the number of samples of healthy subjects that are determined to be negative divided by the total number of samples of healthy subjects. The sensitivity is the proportion of samples of patients that are determined to be positive, i.e., the number of samples of patients that are determined to be positive divided by the total number of samples of patients. Calculate the sensitivity and the specificity when each of the proteins to be tested is used as an antigen for sample testing. The results are shown in Table 2. " / " indicates that the corresponding protein was not obtained, and the measurement was not performed.

[0034] Table 2: Comparison table of detection results of each candidate protein

[0035] The results show that the sensitivity of each potential protein or protein fragment alone in detecting acute myocardial infarction is low, and the clinical application is difficult.

[0036] 7. Analyze the amino acid sequences and structures of the whole proteins of cTnl, HSP65 and VEGFRl. After a large number of preliminary tests, select different sequence fragments, and determine the sensitivity and specificity of detecting acute myocardial infarction according to the method of step 6. The selected sequence fragments and detection results are shown in Table 3.

[0037] Table 3 Comparison of protein fragments and detection results

[0038] The results show that compared with the whole protein and other protein fragments, the sensitivity of cTnl (31-164) is significantly improved while maintaining high specificity. The sensitivity of HSP65 (187-375) is slightly lower than that of HSP65 whole protein and HSP65 (1-540), but the specificity is significantly improved, which can reduce the possibility of false positives while ensuring the detection rate, so HSP65 (187-375) is selected as the preferred fragment. The specificity of VEGFRl (781-1338) and VEGFRl (27-242) is comparable, and the sensitivity detection values are not significantly different, but the AUC area of VEGFRl (781-1338) is larger, which can reduce the possibility of false positives, so VEGFRl (781-1338) is selected as the preferred fragment.

[0039] 8. Use each protein fragment in Table 3 to detect 96 cases of confirmed coronary heart disease patients and 96 cases of healthy controls with the same age and gender as the coronary heart disease cases using indirect enzyme-linked immunoassay (detection method steps 1-6, only the sample is replaced with the sample in this step). The coronary heart disease patients include 50 males and 46 females, aged 40-79 years, with an average age of 54 years. The diagnosis method of the patients conforms to the clinical diagnosis standard of coronary heart disease of the World Health Organization (WHO). The samples of the coronary heart disease patients were collected immediately after the diagnosis was confirmed.

[0040] The results show that compared with the serum reaction of normal people, compared with the whole protein and other protein fragments, the antibodies produced by cTnl (31-164), HSP65 (187-375) and VEGFRl (781-1338) can respectively induce more obvious antigen-antibody reaction in the serum (manifested as a significant increase in OD value). It is proved that these protein fragments have the potential for screening and detecting coronary heart disease.

[0041] Based on steps 6-8, the final selected candidate proteins (fragments) are shown in Table 4.

[0042] Table 4 Comparison of candidate proteins (fragments) and detection results

[0043] Example 3: Effect of protein composition in detecting acute myocardial infarction samples 1. Using the "double index" method, two protein antigens are used for detection.

[0044] The "double index" method refers to: when a certain sample to be tested is detected using a certain antigen combination, if the detection signals of the two antigens in the antigen combination are both positive detection signals, the detection result of the sample is positive, otherwise it is negative. The rest of the operation and the definition of sensitivity and specificity are the same as in Example 2. The results are shown in Table 5.

[0045] Table 5: Comparison of double-antigen combination detection results

[0046] The results show that: when using the "double index" method to detect acute myocardial infarction with double-antigen combinations, the specificity is very high, and the problem of false positives can be basically avoided, but the positive detection rate is low, and the sensitivity is not ideal. Among them, the sensitivity of combinations 2, 6, 9, 17, 22 and 25 is relatively higher, and these combinations are selected for further experiments.

[0047] 3. Increase the protein in the antigen combination, and detect the specificity and sensitivity of the antigen combination according to the "double index" method of step 2 (in this step, if two or more antigen detection signals are positive, it is judged as positive, otherwise it is negative), using the samples and sensitivity and specificity determination method of Example 2. The results are shown in Table 6. It should be noted that the inventors obtained the antigen combinations shown in Table 6 after a large number of preliminary experiments, not just the combinations in Table 6. Due to space limitations, only some of the combinations with better results are selected here.

[0048] Table 6: Comparison of antigen combination detection results of acute myocardial infarction

[0049] The results show that: combinations 1-13 can significantly improve the detection rate and sensitivity while maintaining high specificity, effectively improving the clinical application value.

[0050] Example 4: Single protein detection of drug-induced myocardial damage samples

[0051] The present application also found that cTnI(31-164) and HSP65(187-375) are closely related to drug-induced (especially anti-tumor related drugs) myocardial damage, and are expected to be used for monitoring and detecting drug-induced myocardial damage.

[0052] The existing data shows that the cTnT level significantly increases from 4.6±1.4 pg / mL to 21.3±14.4 pg / mL (p<0.001) during 79 to 146 days after the start of the use of the tumor drug, and then gradually decreases. The high and low values of the ELSIA detection value (enzyme marker 450 nm wavelength) are used to evaluate the change level of the corresponding antibody during the use of the tumor drug (such as doxorubicin, trastuzumab, rituximab, etc.). The collected cases are tracked and detected for 100 days according to the experimental method of Example 2. The results are shown in Tables 7-10 (the cases used in Tables 7-10 are the same).

[0053] Table 7 Detection of ELISA value change results during the use of tumor drugs Table 1

[0054] Table 8 Detection of ELISA value change results during the use of tumor drugs Table 2

[0055] Table 9 Detection of ELISA value change results during the use of tumor drugs Table 3

[0056] Table 10 Detection of ELISA value change results during the use of tumor drugs Table 4

[0057] The results show that as the number of days of use of the tumor drug increases, the levels of the corresponding antibodies of cTnI (31-164) and HSP65 (187-375) significantly increase. Among them, cTnI (31-164) increases significantly at about 30 days, which is about 40 days earlier than cTnI full protein (which increases at about 70 days); HSP65 (187-375) significantly increases at about 20 days, which is about 30 days earlier than HSP65 (which increases at about 50 days); which fully illustrates that the specific protein fragments and antibody binding provided by the present application are stronger, and the detection sensitivity is better. The protein fragments provided by the present application can detect myocardial damage caused by drugs such as tumor drugs.

[0058] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications, variations, modifications, and replacements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A cTnl (31-164) protein fragment, characterized in that: The amino acid sequence of the cTnI (31-164) protein fragment is shown as SEQ ID NO:

1.

2. An antigen combination, characterized by: The antigen combination comprises a HSP65 (187-375) protein fragment, a TNNC1 protein fragment, a DCD (20-110) protein fragment and a NPY (29-97) protein fragment; The amino acid sequence of the HSP65 (187-375) protein fragment is shown as SEQ ID NO: 5, the amino acid sequence of the TNNC1 full protein is shown as SEQ ID NO: 3, the amino acid sequence of the DCD (20-110) protein fragment is shown as SEQ ID NO: 6, and the amino acid sequence of the NPY (29-97) protein fragment is shown as SEQ ID NO:

8.

3. A protein fragment of HSP65 (187-375) characterized in that: The amino acid sequence of the HSP65 (187-375) protein fragment is shown as SEQ ID NO:

5.

4. Use of the cTnI (31-164) protein fragment of claim 1 or the antigen combination of claim 2 or the HSP65 (187-375) protein fragment of claim 3 in the preparation of a product for detecting / identifying acute myocardial infarction.

5. Use of the cTnI (31-164) protein fragment of claim 1 or the antigen combination of claim 2 or the HSP65 (187-375) protein fragment of claim 3 in the preparation of a product for detecting / identifying drug-induced myocardial injury.

6. A reagent or kit for detecting / identifying acute myocardial infarction prepared by using the cTnI (31-164) protein fragment of claim 1 or the antigen combination of claim 2 or the HSP65 (187-375) protein fragment of claim 3.

7. A reagent or kit for detecting / identifying drug-induced myocardial injury prepared by using the cTnI (31-164) protein fragment of claim 1 or the antigen combination of claim 2 or the HSP65 (187-375) protein fragment of claim 3.

8. A reagent or kit for detecting / identifying acute myocardial infarction comprising the cTnI (31-164) protein fragment of claim 1 or the antigen combination of claim 2 or the HSP65 (187-375) protein fragment of claim 3.

9. A reagent or kit for detecting / identifying drug-induced myocardial injury comprising the cTnI (31-164) protein fragment of claim 1 or the antigen combination of claim 2 or the HSP65 (187-375) protein fragment of claim 3.

10. Use according to claim 5, or agent or kit according to claim 7 or 9, wherein: The drug is a drug for treating tumors or cancers. The drug is a drug for treating tumors or cancers.

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