Cardiac troponin I fragment, product prepared from cardiac troponin I fragment and application of cardiac troponin I fragment

Abstract

The invention belongs to the field of biological detection, and particularly relates to a cardiac troponin I fragment, a product prepared from the cardiac troponin I fragment and application of the cardiac troponin I fragment. According to the specific technical scheme, the protein fragment comprises a cTnI (31-164) protein fragment, an HSP65 (187-375) protein fragment and a protein fragment combination. The invention provides a protein antigen or composition for detecting acute myocardial infarction, the detection specificity and sensitivity are ideal, and the protein antigen or composition can be further prepared into related reagents or kits according to needs.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to cardiac troponin I fragments and products prepared therefrom and applications. Background Art

[0002] Acute myocardial infarction (AMI) is the most urgent acute coronary syndrome. It is mainly caused by the rupture of atherosclerotic plaques in the coronary artery, which leads to the exposure of a large amount of thrombogenic substances, followed by acute obstruction of the coronary artery lumen by agglutinated platelets, fibrin and red blood cells. The morbidity and mortality of acute myocardial infarction remain high.

[0003] At present, biochemical detection has become one of the important means of clinically detecting acute myocardial infarction. The biomarkers that have been developed include serum glutamate acetate transferase / aspartyl transferase (SGOT / AST), lactate dehydrogenase (LDH), myoglobin, creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB) and troponin, etc. However, the sensitivity and specificity of these markers need to be improved, and the available biomarkers are relatively limited.

[0004] Therefore, if the optional range of biomarkers can be expanded and the sensitivity and specificity of detecting acute myocardial infarction can be improved, it will have great application prospects. Summary of the invention

[0005] The purpose of the present invention is to obtain cardiac troponin I fragments and products prepared therefrom and their application.

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

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

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

[0009] Correspondingly, the HSP65 (187-375) protein fragment, the amino acid sequence of the HSP65 (187-375) protein fragment is shown in 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 in the preparation of products for detecting / identifying acute myocardial infarction and / or drug-induced myocardial injury.

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

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

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

[0014] The present invention has the following beneficial effects: The present invention provides antigens for detecting autoantibodies related to acute myocardial infarction / myocardial injury: cTnI (31-164), HSP65 (187-375), which can be used to detect the presence of autoantibodies against the above antigen combination in biological samples from subjects, thereby determining whether the subject suffers from acute myocardial infarction / myocardial injury or has a potential risk of suffering from acute myocardial infarction / myocardial injury. Compared with the whole cTnI protein and other protein fragments of cTnI, the sensitivity of cTnI (31-164) is significantly improved while maintaining a high specificity, and the clinical application value is also significantly improved. Although the sensitivity of HSP65 (187-375) is slightly lower than that of the whole HSP65 protein and HSP65 (1-540), the specificity is significantly improved, which can reduce the possibility of false positives while ensuring the detection rate. Therefore, cTnI (31-164) and HSP65 (187-375) can be used alone or in combination with other biomarkers to detect / diagnose acute myocardial infarction, can be used to detect myocardial infarction and chronic myocardial damage (such as myocardial loss caused by anti-tumor drugs), and can also be used to assist in the identification of coronary heart disease and guide the use of clinical drugs. The biological sample can be serum, plasma, whole blood, saliva, oral mucosal swab, urine, lymph, cerebrospinal fluid, etc. According to the specific situation, the biological sample can be pre-treated by extraction, dilution, enrichment and other means, and the use method is diverse and easy to operate. DETAILED DESCRIPTION

[0015] The present invention discovers for the first time that the cTnI (31-164) protein fragment is closely related to acute myocardial infarction and myocardial injury. The cTnI (31-164) protein fragment is used to detect acute myocardial infarction with extremely high specificity and ideal sensitivity. It can be used alone or in combination with other biomarkers to prepare products (such as kits) related to the detection and diagnosis of acute myocardial infarction.

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0017] Example 1: Construction, expression and purification of recombinant vectors of antigens The method for obtaining the antigen can be: synthesizing the coding DNA of the protein antigen, using the synthetic DNA as a template, designing primers, cloning the gene fragment of the protein antigen or its fragment into the expression plasmid by molecular cloning means such as PCR, enzyme digestion, and ligation, and then expressing it through Escherichia coli, yeast or cells, and then purifying it by chromatography to obtain the target protein. At the same time, 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 it will not essentially change the binding characteristics of the antigen with its own antibody.

[0018] Potential proteins related to acute myocardial infarction detection were selected, and their database IDs are shown in Table 1.

[0019] Table 1 Comparison table of potential protein database IDs

[0020] Using human cDNA library (purchased from Invitrogen) or whole gene synthetic DNA as template, primers are designed respectively, and the gene fragment of the protein is cloned into pET28 plasmid by molecular cloning methods such as PCR, enzyme digestion, and ligation. At the same time, HIS, FLAG and other tags are added to the N-terminus of the protein to form a fusion protein. The obtained recombinant expression vector is identified by DNA sequencing to confirm that it contains the correct protein gene fragment. It should be noted that the addition of tags is only for the convenience of identifying and extracting proteins, and does not have a decisive effect on the function of the protein as an antigen. When used, no tags are added or other tags are added as needed or other marking / identification methods are adopted.

[0021] The recombinant plasmid containing the protein gene fragment was transformed into the competent cells of E. coli BL21 (DE3), and clones were picked and inoculated into LB medium and cultured in a shaking incubator at 37°C. 600 When the pH value was about 0.8, the temperature was lowered to 16°C, 0.1 mM isopropylthio-β-D-galactoside (IPTG) was added to each LB culture medium, and the expression was induced overnight to obtain bacteria.

[0022] The induced expression of the bacteria was collected by centrifugation and rinsed twice with PBS. The bacteria were resuspended and dispersed with lysis solution (5-10 mL lysis solution was added per gram of bacteria), ice bathed, and ultrasonically disrupted (ultrasonic power 200W, disruption 5s, rest 5s). After disruption, centrifuged at 13000rpm, 10℃ for 20 minutes, and the supernatant was taken. After two-step purification by Ni column affinity chromatography and molecular sieve chromatography, SDS-PAGE electrophoresis was used to confirm the molecular weight and purity of the protein. After the concentration was determined by Bradford method, it was stored at -80℃ for standby use. 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; the whole VEGFR1 protein was not obtained.

[0023] Example 2: Demonstration of the effect of each candidate protein alone in detecting acute myocardial infarction samples 1. The solutions and reagents used in this example are as follows: (1) The coating buffer is PBS buffer, pH = 7.4. The preparation method is as follows: accurately weigh 3.58 g Na2HPO4·12H2O, 0.23 g KH2PO4·2H2O, 0.2 g KCl, and 8.0 g NaCl, dissolve them in water, and add water to make up to 1 L.

[0024] (2) Blocking solution / sample diluent / antibody diluent: Dissolve 10 g BSA (bovine serum albumin) in 800 ml coating buffer and adjust the volume to 1 L with coating buffer.

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

[0026] (4) TMB colorimetric reagent, purchased from KPL Company.

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

[0028] 2. Solid-phase coating of the protein to be tested. Dilute the purified candidate proteins obtained in Example 1 to 5 μg / mL with coating buffer, add to a 96-well plate, 50 μL per well, and coat overnight at 4°C. Pour out the solution the next day, spin dry, and wash three times with washing solution, 200 μL per well each time. Then add 200 μL of blocking solution to each well, incubate at room temperature for 1 hour, pour out the blocking solution, spin dry, and wash three times with washing solution, 200 μL per well each time, and spin dry again; obtain the solid-phase coated antigen in the 96-well plate.

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

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

[0031] 5. Color development and optical density reading. After step 4, add 50 μL of TMB color developer to each well of the 96-well plate, shake for 15 seconds, react in the dark at room temperature for 15 minutes, and then add 50 μL of stop solution; then use an ELISA reader to read the absorbance at a wavelength of 450 nm to obtain the detection signal (S) of each sample to be tested.

[0032] 6. Sensitivity and specificity analysis. 384 positive samples (serum from patients diagnosed with myocardial infarction) and 384 negative samples (serum from healthy subjects) were taken respectively, and the detection signal (S) of each sample was determined according to the above method (absorption value at a wavelength of 450nm). The negative samples were taken as negative reference samples, and the mean (M) and standard deviation (SD) of the detection signals (S) of all negative reference samples were calculated, with M+3SD as the Cut Off value. Samples with a detection signal (S) ≥ Cut Off value (S≥M+3SD) were defined as positive; samples with a detection signal (S) < Cut Off value (S<M+3SD) were defined as negative.

[0033] Specificity and sensitivity were calculated based on the positive and negative results of the samples. Specificity refers to the proportion of samples from healthy subjects that were correctly judged as negative, that is, the number of samples judged as negative in the negative samples divided by the total number of negative samples. Sensitivity refers to the proportion of samples from positive patients enrolled in the group that were judged as positive, that is, the number of samples judged as positive in the positive samples divided by the total number of positive samples. The sensitivity and specificity of sample testing using each tested protein as an antigen were calculated. The results are shown in Table 2. " / " indicates that the corresponding protein was not obtained and was not measured.

[0034] Table 2 Comparison table of detection results of candidate proteins

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

[0036] 7. Analyze the amino acid sequence and structure of the whole protein of cTnI, HSP65 and VEGFR1. After a large number of preliminary tests, different sequence fragments were selected, and the sensitivity and specificity of detecting acute myocardial infarction were determined according to the method in step 6. The selected sequence fragments and the test results are shown in Table 3.

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

[0038] The results showed that compared with the whole protein and other protein fragments, the sensitivity of cTnI (31-164) was significantly improved while maintaining a high specificity. Although the sensitivity of HSP65 (187-375) was slightly lower than that of HSP65 whole protein and HSP65 (1-540), its specificity was significantly improved, which could reduce the possibility of false positives while ensuring the detection rate. Therefore, HSP65 (187-375) was selected as the preferred fragment. The specificity results of VEGFR1 (781-1338) and VEGFR1 (27-242) were similar, and the sensitivity detection values ​​were not much different, but the AUC area of ​​VEGFR1 (781-1338) was larger, which could reduce the possibility of false positives. Therefore, VEGFR1 (781-1338) was selected as the preferred fragment.

[0039] 8. Using the protein fragments in Table 3, 96 patients diagnosed with coronary heart disease and 96 healthy controls of the same age and gender as the coronary heart disease cases were tested by indirect enzyme-linked immunosorbent assay (the detection method is the same as steps 1-6, except that the samples are replaced by the samples in this step). The coronary heart disease patients included 50 males and 46 females, with an age range of 40-79 years old and an average age of 54 years old. The diagnostic method for the patient's diagnosis was in accordance with the World Health Organization (WHO) clinical diagnostic criteria for coronary heart disease. Samples were collected immediately from patients with coronary heart disease when the diagnosis was confirmed.

[0040] The results showed that compared with the serum reaction of normal people, the antibodies produced by cTnI (31-164), HSP65 (187-375) and VEGFR1 (781-1338) could induce more obvious antigen-antibody reaction in serum (manifested by a significant increase in OD value) compared with the whole protein and other protein fragments, proving that these protein fragments have the potential to screen and detect coronary heart disease.

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

[0042] Table 4 Comparison table of candidate proteins (fragments) and test results Antigen corresponding to the antibody name Sensitivity Specificity Sequence number cTnI(31-164) 56.25 89.58 SEQ ID NO.1 HBEGF(19-160) 33.33 91.76 SEQ ID NO.2 TNNC1 33.33 93.75 SEQ ID NO.3 NPPA(26-151) 25 97.92 SEQ ID NO.4 HSP65(187-375) 41.67 89.58 SEQ ID NO.5 DCD(20-110) 35.42 95.83 SEQ ID NO.6 VEGFR1(781-1338) 43.75 97.92 SEQ ID NO.7 NPY(29-97) 50 91.67 SEQ ID NO.8

[0043] Example 3: Demonstration of the effect of protein combination combined with detection of acute myocardial infarction samples 1. Use the "dual indicator" method and two protein antigens for detection.

[0044] The "double index" method means that when a sample to be tested is tested using a certain antigen combination, if the detection signals obtained by 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 remaining operations and the definitions of sensitivity and specificity are the same as those in Example 2. The results are shown in Table 5.

[0045] Table 5 Comparison table of double antigen combination test results

[0046] The results showed that when using the "double index" method and double antigen combination to detect acute myocardial infarction, the specificity was extremely high and the problem of false positives could be basically avoided, but the positive detection rate was low and the sensitivity was not ideal. Among them, the sensitivity of combinations 2, 6, 9, 17, 22 and 25 was relatively higher, and these combinations were selected to continue the subsequent experiments.

[0047] 3. Increase the protein in the antigen combination, and according to the "double index" method of step 2 (in this step, if there are two or more antigen detection signals as positive detection signals, it is judged as positive, otherwise it is negative), use the sample and sensitivity and specificity determination method of Example 2 to detect the specificity and sensitivity of the antigen combination. 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, rather than only testing the combinations in Table 6. Due to space limitations, only some combinations with better effects are selected here.

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

[0049] The results showed that combinations 1-13 could significantly improve the detection rate and sensitivity while maintaining high specificity, effectively enhancing the clinical application value.

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

[0051] The present invention also found that cTnI (31-164) and HSP65 (187-375) are closely related to myocardial damage caused by drugs (especially anti-tumor related drugs), and are expected to be used to monitor and detect myocardial damage caused by drugs.

[0052] Existing data show that cTnT levels increased significantly from 4.6±1.4pg / mL to 21.3±14.4pg / mL (p<0.001) during the period from 79 to 146 days after the start of use of tumor drugs, and then gradually decreased. The level of changes in the corresponding antibodies during the use of tumor drugs (such as doxorubicin, trastuzumab, rituximab, etc.) was evaluated using the high and low ELS IA detection values ​​(microplate reader measuring 450nm wavelength). The collected cases were followed up 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 Results of ELISA value changes during the use of tumor drugs Table 1

[0054] Table 8 Results of changes in ELISA values ​​during the use of tumor drugs Table 2

[0055] Table 9 Results of ELISA value changes during the use of tumor drugs Table 3

[0056] Table 10 Results of ELISA value changes during the use of tumor drugs Table 4

[0057] The results showed that as the number of days of use of tumor drugs increased, the corresponding antibody levels of cTnI (31-164) and HSP65 (187-375) increased significantly. Among them, cTnI (31-164) increased significantly at around 30 days, about 40 days earlier than the cTnI whole protein (increased at around 70 days); HSP65 (187-375) increased significantly at 20 days, about 30 days earlier than HSP65 (increased at around 50 days); it fully demonstrated that the specific protein fragments provided by the present invention have a stronger ability to bind to antibodies and better detection sensitivity. The protein fragments provided by the present invention can detect myocardial damage caused by drugs such as tumor drugs.

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

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

1.

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

8.

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

5.

4. Use of the cTnI (31-164) protein fragment of claim 1, 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, 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 using the cTnI (31-164) protein fragment of claim 1, 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 using the cTnI (31-164) protein fragment of claim 1, 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, 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, the antigen combination of claim 2, or the HSP65 (187-375) protein fragment of claim 3.

10. The use according to claim 5, or the reagent or kit according to claim 7 or 9, characterized in that: The medicine is a medicine for treating tumors or cancer.

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