CN111879947A

CN111879947A - Application of specific protein WIPI1 in early myocardial infarction diagnosis reagent and kit

Info

Publication number: CN111879947A
Application number: CN202010739518.2A
Authority: CN
Inventors: 李觉
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2020-07-28
Filing date: 2020-07-28
Publication date: 2020-11-03

Abstract

The invention relates to an application of a specific protein WIPI1 in an early myocardial infarction diagnostic reagent, wherein the amino acid sequence of the specific protein WIPI1 is shown in SEQ ID NO.1, and the content of the specific protein WIPI1 in peripheral blood is increased after myocardial injury during specific detection. Compared with the prior art, the diagnostic reagent of the technical scheme of the invention is beneficial to early diagnosis, can reflect the severity of myocardial damage, can be used as an index for early myocardial infarction diagnosis and prediction, has a simple and easy-to-operate detection method, better detection sensitivity and smaller system error, and can widely popularize the application of specific protein WIPI1 coded by WIPI1 gene in the myocardial damage diagnostic reagent; the kit has the advantages of simple detection method, low cost, direct and reliable detection result, and is suitable for large-scale screening and diagnosis.

Description

Application of specific protein WIPI1 in early myocardial infarction diagnosis reagent and kit

Technical Field

The invention relates to the field of biomedicine, in particular to application of a specific protein WIPI1 in an early myocardial infarction diagnosis reagent and a kit.

Background

Currently, the main biochemical markers for detecting Acute Myocardial Infarction (AMI) include creatine phosphokinase isozyme (CK-MB), Lactate Dehydrogenase (LDH), cardiac troponin (cTnT), cardiac troponin I (cTnI) and the like. CK-MB, LDH are widely distributed in the body and thus have low specificity, and many diseases can cause the increase of them and have short maintenance time in the blood; cTnT has high homology with skeletal muscle troponin t (stnt), and cross-reaction easily occurs. The cTnI has the advantages of high specificity, early appearance time, high sensitivity, long duration in blood and the like, and can be used as an early AMI diagnosis index, so the current kit detection mode mainly surrounds cardiac troponin I (cTnI).

The detection method of cardiac troponin I (cTnI) comprises enzyme-linked immunosorbent assay (ELISA) immunoassay paper strip detection and the like. Although the specificity of cardiac troponin i (ctni) is higher, its predictability for myocardial injury is not high. The immunoassay test strip is used for detection, the method mainly adopts the principle of a colloidal gold immunochromatography method or a fluorescence immunochromatography method, and the method is simple to operate, convenient, fast, low in sensitivity, low in accuracy and prone to detection omission.

CN1227533C uses the specific expression of human cardiac calcium binding protein S100A1 in the plasma of patients with myocardial infarction, the marked human cardiac calcium binding protein S100A1 monoclonal or polyclonal antibody is fixed on a support as a detection reagent, and the content of the antigen-cardiac calcium binding protein S100A1 in the blood is measured according to the color change after the reaction of the detection reagent and the antigen in a sample by an ELISA method or a gold-labeling method, so as to draw a diagnosis conclusion. The method is still to be improved in accuracy and is easy to miss detection.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the application of the specific protein WIPI1 in the early myocardial infarction diagnosis reagent and the kit, and the detection of the content of WIPI1 can reflect the severity of myocardial damage, so that the severity can be used as an index for early myocardial infarction diagnosis and prediction.

The purpose of the invention can be realized by the following technical scheme:

the specific protein WIPI1 has better application in early myocardial infarction diagnostic reagents, wherein the specific protein WIPI1 is coded by a WIP1 gene, and the amino acid sequence of the specific protein WIPI1 is shown as SEQ ID NO.1 in the attachment. Wherein the WIPI1 gene is derived from human.

Further, the amount of specific protein WIPI1 in peripheral blood increased after myocardial injury. The WIPI1 specific protein can be used as an important protein molecular marker for detecting the occurrence of diseases in the early stage of myocardial infarction.

Further, the content of specific protein WIPI1 in peripheral blood was obtained by ELISA.

Further, the immunoadsorption assay described herein used WIPI1 as an antigen.

Furthermore, the concentration of WIPI1 used in the immunoadsorption experiment is 10-20 mu g/ml.

Further, the concentration of the specific protein WIPI1 was obtained by measuring absorbance in the immunoadsorption assay using a microplate reader.

Further, the content of specific protein WIPI1 in peripheral blood is detected by a WIPI1 activity detection kit.

Further, the WIPI1 activity detection kit adopts spectrophotometry to detect the activity of Wip1 enzyme or the activity of purified WIPI1 enzyme in peripheral blood cells or tissue lysate, so as to obtain the change trend of the content of specific protein WIPI 1.

Further, an absorption peak at 405nm was selected for spectrophotometric detection.

The kit can be prepared by the technical scheme of the invention, and the prepared kit comprises a solid phase carrier coated with WIPI1 antigen and enzyme-labeled WIPI1 antigen.

Compared with the prior art, the invention has the following advantages:

1) the diagnostic reagent of the technical scheme of the invention obviously improves the efficiency of early myocardial infarction diagnosis, can reflect the severity of myocardial damage, is used as an index for early myocardial infarction diagnosis and prediction, has simple and easy-to-operate detection method, better detection sensitivity, smaller system error and can avoid the condition of missed detection, and can widely popularize the application of specific protein WIPI1 coded by WIP1 gene in the myocardial damage diagnostic reagent.

2) The kit has the advantages of simple detection method, low cost, direct detection result, stable and reliable result, and is suitable for large-scale screening and diagnosis.

Drawings

FIG. 1 is a schematic diagram of an experiment in the present invention;

FIG. 2 is a schematic diagram showing the effect of detecting infarction 1 hour after myocardial infarction in an animal experiment according to the present invention;

FIG. 3 is a graph comparing the proteins of the present invention with significant increases in myocardial infarction occurring at 10 minutes and 1 hour;

FIG. 4 is a functional cluster analysis graph of proteins significantly elevated in 1 hour after myocardial infarction in the present invention;

FIG. 5 is an electron microscope result showing that significant autophagosomes were seen in myocardial tissue after 1 hour of premature myocardial infarction in accordance with the present invention;

FIG. 6 is a graph showing the changes of the relevant genes occurring in 10 minutes and 1 hour after myocardial infarction in the present invention;

FIG. 7 is a graph comparing the changes in WIPI1 protein occurring at 10 minutes and 1 hour after myocardial infarction in the present invention.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Examples

The experimental model in this example is the detection of protein changes within the very early (1 hour) phase of myocardial infarction by DIA-MS proteome sequencing, and is shown in figure 1.

Animal experiment modeling: in the invention, in order to search for proteins and genes which are obviously changed in the myocardial infarction process, a myocardial infarction model is constructed by using a model mouse c 57. The myocardial infarction model is first constructed by ligating the left anterior descending branch of the coronary artery. The effect after ligation is shown in fig. 2, the infarction effect is detected 1 hour after myocardial infarction, and a large area of the apical area is whitish, which suggests that the tissue is in large area ischemia, thus proving successful infarction experiment.

And (3) detection finding: at 10min myocardial infarction, 12 genes with increased expression were found in this example, while after 1h, a significantly changed gene increased to 22, with UBQLN1 being expressed at both times (see fig. 3). Then, this example performed cluster analysis on the genes of 1h, and as a result, it was found that these genes were closely related to autophagy of cells (see FIG. 4).

It was also found by electron microscopy that autophagic vesicles were found in this example in 1h of myocardial infarction tissue (see FIG. 5). Previous studies have shown that autophagy may play a protective role in the myocardial infarction, and the results of this example show that autophagy may have already functioned just as the myocardial infarction occurred. Moreover, among these altered genes, RRAD and WIPI1 were found to be specifically expressed in cardiac tissue and to be potential markers for very early diagnosis of myocardial infarction. Quantitative PCR detection revealed that there was no significant change in the expression of these two genes at this time (see figure 6), whereas the protein expression of WIPI1 was significantly elevated at 1h (see figure 7).

The above results in this example show that very early myocardial infarction may be significantly associated with autophagy, whereas WIPI1 may be a potential target for very early diagnosis of myocardial infarction.

The kit can be prepared by the technical scheme of the invention, comprises a solid phase carrier coated with WIPI1 antigen and enzyme-labeled WIPI1 antigen, and the specific detection process of the kit is as follows:

the content of the specific protein WIPI1 in peripheral blood is obtained by an enzyme-linked immunosorbent assay, wherein WIPI1 is adopted as an antigen in the enzyme-linked immunosorbent assay, the concentration of the used WIPI1 is 10-20 mu g/ml, and the absorbance is tested by an enzyme-linked immunosorbent assay to obtain the concentration of the corresponding specific protein WIPI 1.

a. Envelope antigens

1) The antigen was dissolved in 50mM carbonate coating buffer (pH 9.6) to a concentration of 10-20. mu.g/ml, and 100. mu.l/well was applied to a 96-well microplate and left overnight at 4 ℃.

2) The next day, the coating solution was discarded, and washed 3 times with PBST, and 150. mu.l of 1% BSA was added to each well and blocked at 37 ℃ for 1 hour.

3) After 3 PBST washes, 100. mu.l of serum was added at different fold-rate dilutions to each well, and a control sample was added and incubated for 2 hours at 37 ℃.

4) After PBST was washed 5 times, 100. mu.l of diluted secondary HRP-labeled antibody was added and incubated at 37 ℃ for 1 hour.

5) After PBST is washed for 5 times, the color developing agent develops color for 20min, and the A405 absorption value is read on an enzyme-linked immunosorbent assay.

b. Coating cells

1) The 96-well culture plate was seeded with 1X 104cells/well and cultured overnight at 37 ℃.

2) The following day the plates were washed 2-3 times with PBS.

3) 125. mu.l/well of 10% Forma lin (1:10 dilution) were added and fixed for 15min at room temperature.

4) By ddH₂The plates were washed 3 times with O, air dried and stored at 2-8 ℃ until use.

5) Washed 3 times with PBST and blocked by adding 150. mu.l of 1% BSA per well for 1 hour at 37 ℃.

6) After 3 PBST washes, 100. mu.l of serum was added at different fold-rate dilutions to each well, and a control sample was added and incubated for 2 hours at 37 ℃.

7) After PBST was washed 5 times, 100. mu.l of diluted secondary HRP-labeled antibody was added and incubated at 37 ℃ for 1 hour.

8) After PBST is washed for 5 times, the color developing agent develops color for 20min, and the A405 absorption value is read on an enzyme-linked immunosorbent assay. 8) After PBST is washed for 5 times, the color developing agent develops color for 20min, and the A405 absorption value is read on an enzyme-linked immunosorbent assay.

9) And (3) taking the OD value of the absorbance as a vertical coordinate (Y) and the concentration of the corresponding substance to be detected as a horizontal coordinate (X) to obtain a corresponding curve, and converting the content of the substance to be detected of the sample into the corresponding concentration from the standard curve according to the OD value of the substance to be detected.

Finally, the corresponding relation between the myocardial damage degree and the detection concentration range of the specific protein WIPI1 is determined by experiments, so that the myocardial damage condition of the subject can be judged.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

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Claims

1. An application of a specific protein WIPI1 in an early myocardial infarction diagnostic reagent is characterized in that the amino acid sequence of the specific protein WIPI1 is shown as SEQ ID NO. 1.

2. The use of the specific protein WIPI1 in the diagnosis of early myocardial infarction as claimed in claim 1, wherein the content of the specific protein WIPI1 in peripheral blood is increased after myocardial damage.

3. The use of the specific protein WIPI1 in the reagent for diagnosing early myocardial infarction as claimed in claim 2, wherein the content of the specific protein WIPI1 in the peripheral blood is obtained by ELISA.

4. The use of the specific protein WIPI1 in the diagnosis of early myocardial infarction as claimed in claim 3, wherein the immunoadsorption assay uses WIPI1 as antigen.

5. The use of the specific protein WIPI1 in the early myocardial infarction diagnostic reagent according to claim 3, wherein the concentration of the WIPI1 used in the immunoadsorption test is 10-20 μ g/ml.

6. The use of the specific protein WIPI1 in the reagent for diagnosing early myocardial infarction as claimed in claim 3, wherein the concentration of the specific protein WIPI1 is obtained by measuring the absorbance through an enzyme-labeling instrument in the immunoadsorption experiment.

7. The use of the specific protein WIPI1 in the reagent for diagnosing early myocardial infarction as claimed in claim 2, wherein the content of the specific protein WIPI1 in the peripheral blood is detected by a WIPI1 activity detection kit.

8. The use of the specific protein WIPI1 in the reagent for diagnosing early myocardial infarction as claimed in claim 7, wherein the WIPI1 activity assay kit employs spectrophotometry to detect the activity of WIPI1 enzyme or the activity of purified WIPI1 enzyme in peripheral blood cells or tissue lysates, so as to obtain the variation trend of the content of the specific protein WIPI 1.

9. The use of the specific protein WIPI1 in the preparation of diagnostic reagents for early myocardial infarction as claimed in claim 8, wherein the absorbance peak at 405nm is measured spectrophotometrically.

10. A kit comprising a solid support coated with WIPI1 antigen, an enzyme-labeled WIPI1 antigen.