Biomarker as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: detecting the concentration of cytoplasmic acyl-coenzyme A thioester hydrolase in a sample by adopting an ELISA (Enzyme-Linked Immunosorbent Assay) method, and carrying out regression analysis through Logistic, the correlation between the serum level of the cytoplasm acyl-coenzyme A thioester hydrolase in an AD (Alzheimer's disease) patient and a vascular dementia (VD) patient and the cognitive impairment degree and the A beta 40 content is evaluated, and whether the expression of the cytoplasm acyl-coenzyme A thioester hydrolase is closely related to the AD progress or not is judged. An effective molecular marker is provided for differential diagnosis of an AD patient and a VD patient, a large number of tests prove that the cytoplasm acyl-coenzyme A thioester hydrolase is highly expressed in the AD patient, the cytoplasm acyl-coenzyme A thioester hydrolase serving as an AD serum molecular marker has high diagnostic value, and when the serum content of the cytoplasm acyl-coenzyme A thioester hydrolase is higher than 62.5 pg / ml, the cytoplasm acyl-coenzyme A thioester hydrolase can assist AD diagnosis and can also serve as an auxiliary index for distinguishing AD and VD. The kit is applied to preparation of a kit for differential diagnosis of AD and VD.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and particularly relates to a biomarker and its preparation method and application. Background Art

[0002] There are many diseases that can cause dementia clinically. Alzheimer's disease (AD) and vascular dementia (VD) are the most common, accounting for about 90%. Pathological biopsy and autopsy are the only ways to confirm the diagnosis, but they are not very helpful for the specific implementation of clinical work. Each of them has its own characteristics in clinical manifestations, but it is often difficult to distinguish them in actual clinical work.

[0003] Although there are multiple diagnostic criteria and methods clinically for diagnosing and differentiating VD patients from AD patients, it is difficult to strictly separate these two diseases, which cannot meet the needs of staff, and the diagnosis and differentiation lack sensitivity, specificity, and accuracy. Therefore, there is an urgent need to find a method that can assist in the differential diagnosis of AD patients and VD patients. Summary of the Invention

[0004] To solve the above technical problems, a biomarker and its preparation method and application are provided. This technical solution solves the problem that although there are multiple diagnostic criteria and methods clinically for diagnosing and differentiating VD patients from AD patients, it is difficult to strictly separate these two diseases, which cannot meet the needs of staff, and the diagnosis and differentiation lack sensitivity, specificity, and accuracy as mentioned in the above background art.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a biomarker is provided, and the biomarker is cytoplasmic acyl-CoA thioester hydrolase.

[0006] In the second aspect of the present invention, a preparation method of a biomarker is further provided, including: Extracting mRNA from a specific biological tissue, such as the brain, obtaining cDNA through reverse transcription, amplifying the cDNA sequence encoding cytoplasmic acyl-CoA thioester hydrolase using primers, and cloning the amplified cDNA sequence into an expression vector; Using a prokaryotic expression system such as Escherichia coli or a eukaryotic expression system such as mammalian cells to express the cloned gene; Optimizing the expression level of the protein through induced expression and under appropriate culture conditions; Using appropriate purification techniques to isolate and purify the expressed protein, and the biomarker can be obtained.

[0007] Preferably, the inducible expression is the expression in Escherichia coli induced by IPTG.

[0008] Preferably, the purification technique includes any one of affinity chromatography and ion exchange chromatography.

[0009] In the second aspect of the present invention, there is also provided an application of a biomarker, including: Detecting the concentration of cytoplasmic acyl-CoA thioester hydrolase in a sample by ELISA method, and performing regression analysis by Logistic to evaluate the correlation between the serum level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) and Aβ 40 content in AD patients and VD patients, and determining whether the expression of cytoplasmic acyl-CoA thioester hydrolase is closely related to the AD disease process; Judging the sensitivity, specificity and accuracy of cytoplasmic acyl-CoA thioester hydrolase as a biomarker in AD patients and VD patients by ROC curve analysis; Combining cytoplasmic acyl-CoA thioester hydrolase with ALG-2-interacting protein X to prepare an AD auxiliary diagnostic kit.

[0010] Preferably, there is a significant positive correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and Aβ 40 content, indicating that cytoplasmic acyl-CoA thioester hydrolase is involved in the metabolism of Aβ 40 metabolism.

[0011] Preferably, in the Logistic regression analysis, there is a significant negative correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) in AD patients, and there is no significant correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) in VD patients.

[0012] Preferably, in the ROC curve analysis, the area under the curve AUC of cytoplasmic acyl-CoA thioester hydrolase for diagnosing AD is 0.83, the optimal diagnostic concentration value is 62.5 pg / mL, its diagnostic sensitivity is 80%, specificity is 74%, and accuracy is 77%.

[0013] Preferably, in the ROC curve analysis, the area under the curve AUC of cytoplasmic acyl-CoA thioester hydrolase for differentiating and diagnosing AD and VD is 0.89, proving that cytoplasmic acyl-CoA thioester hydrolase can effectively distinguish AD patients and VD patients.

[0014] Preferably, for the AD diagnostic kit prepared by combining the cytoplasmic acyl-CoA thioester hydrolase with ALG-2-interacting protein X, its AUC is 0.95, the diagnostic sensitivity is 93%, the specificity is 91%, and the accuracy is 92%.

[0015] Compared with the prior art, the present invention provides a biomarker, its preparation method and application, and has the following beneficial effects: The present invention provides an effective molecular marker for the differential diagnosis of AD patients and VD patients. Through a large number of experiments, the present invention proves that cytoplasmic acyl-CoA thioester hydrolase is highly expressed in AD patients. As an AD serum molecular marker, it has high diagnostic value. When its serum content is higher than 62.5 pg / ml, it can assist in the diagnosis of AD and can also be used as an auxiliary index for differentiating AD and VD. It is applied to the preparation of a kit for differentiating and diagnosing AD and VD, and has sensitivity, specificity and accuracy, meeting the needs of the staff. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the preparation method of the biomarker in the present invention; Figure 2 It is a schematic diagram of the expression of cytoplasmic acyl-CoA thioester hydrolase in the sera of AD patients and normal controls in the present invention; Figure 3 It is a correlation analysis diagram between cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) of AD patients and VD patients in the present invention; Figure 4 It is a ROC curve analysis diagram of the expression of cytoplasmic acyl-CoA thioester hydrolase in the sera of AD patients and normal volunteers in the present invention; Figure 5 It is a ROC curve analysis diagram of the expression of cytoplasmic acyl-CoA thioester hydrolase in the sera of AD patients and VD patients in the present invention; Figure 6 It is for the correlation analysis between serum ACOT7 expression and Aβ 40 content in the present invention; Figure 7 It is a schematic diagram of the ROC curve analysis for diagnosing AD by jointly applying cytoplasmic acyl-CoA thioester hydrolase and ALG-2-interacting protein X in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples, and those skilled in the art can think of other obvious variations.

[0018] Example 1 Please refer to Figures 1 - 7 As shown, in the first aspect of the present invention, a biomarker is provided, and the biomarker is cytoplasmic acyl-CoA thioester hydrolase.

[0019] In the second aspect of the present invention, a method for preparing a biomarker is further provided, including: S101. Extract mRNA from a specific biological tissue, such as the brain, obtain cDNA by reverse transcription, amplify the cDNA sequence encoding cytoplasmic acyl-CoA thioester hydrolase using primers, and clone the amplified cDNA sequence into an expression vector; S102. Use a prokaryotic expression system such as Escherichia coli or a eukaryotic expression system such as mammalian cells to express the cloned gene; S103. Optimize the protein expression level by induced expression and under appropriate culture conditions; S104. Use appropriate purification techniques to isolate and purify the expressed protein, and the biomarker can be obtained.

[0020] The induced expression is to induce the expression in Escherichia coli using IPTG.

[0021] The purification techniques include any one of affinity chromatography and ion exchange chromatography.

[0022] In the second aspect of the present invention, an application of a biomarker is further provided, including: Detect the concentration of cytoplasmic acyl-CoA thioester hydrolase in a sample by ELISA method, and perform regression analysis by Logistic to evaluate the serum level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) and Aβ 40 content in AD patients and VD patients, and judge whether the expression of cytoplasmic acyl-CoA thioester hydrolase is closely related to the AD disease process; Judge the sensitivity, specificity and accuracy of cytoplasmic acyl-CoA thioester hydrolase as a biomarker in AD patients and VD patients by ROC curve analysis; Prepare an AD auxiliary diagnostic kit by combining cytoplasmic acyl-CoA thioester hydrolase with ALG-2-interacting protein X.

[0023] There is an obvious positive correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and Aβ 40 content, indicating that cytoplasmic acyl-CoA thioester hydrolase is involved in the metabolism of Aβ 40 metabolism.

[0024] In the Logistic regression analysis, there was a significant negative correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) in AD patients, indicating that there was no significant correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) in VD patients.

[0025] In the ROC curve analysis, the area under the curve (AUC) of cytoplasmic acyl-CoA thioester hydrolase for diagnosing AD was 0.83, the optimal diagnostic concentration value was 62.5 pg / mL, the diagnostic sensitivity was 80%, the specificity was 74%, and the accuracy was 77%, demonstrating that cytoplasmic acyl-CoA thioester hydrolase could be used to diagnose AD patients and had good diagnostic value.

[0026] In the ROC curve analysis, the area under the curve (AUC) of cytoplasmic acyl-CoA thioester hydrolase for differentiating and diagnosing AD and VD was 0.89, proving that cytoplasmic acyl-CoA thioester hydrolase could effectively distinguish AD patients from VD patients.

[0027] The AD diagnostic kit prepared by combining cytoplasmic acyl-CoA thioester hydrolase and ALG-2-interacting protein X had an AUC of 0.95, a diagnostic sensitivity of 93%, a specificity of 91%, and an accuracy of 92%.

[0028] Example 2 Confirm the high expression of cytoplasmic acyl-CoA thioester hydrolase in the serum of AD patients: In cooperation with the clinic, the present invention obtained 366 serum samples from AD patients and 316 serum samples from age-matched volunteers. The concentration of cytoplasmic acyl-CoA thioester hydrolase in the samples was detected by ELISA method, and the results were as Figure 2 shown. The ELISA results showed that, compared with the normal control, the expression of cytoplasmic acyl-CoA thioester hydrolase in the serum of AD patients was significantly increased, with statistical significance.

[0029] Example 3 Evaluate the correlation between cytoplasmic acyl-CoA thioester hydrolase and the disease process: SPSS 14.0 software was used for Logistic regression analysis to evaluate the correlation between the serum level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score), and to determine whether the expression of cytoplasmic acyl-CoA thioester hydrolase was closely related to the disease process. The specific results were as Figure 3As shown in the figure. The results showed that there was a significant negative correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the MMSE score of AD patients (an important indicator of disease severity) (r = -0.85; p < 0.001). However, there was no significant correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the MMSE score of VD patients (an important indicator of disease severity).

[0030] Example 4 Evaluate the diagnostic value of cytoplasmic acyl-CoA thioester hydrolase as a serum biomarker in the preparation of a kit for the auxiliary diagnosis of AD: Through ROC curve analysis, the sensitivity, specificity, and accuracy of cytoplasmic acyl-CoA thioester hydrolase as a biomarker were judged, and its optimal diagnostic concentration (Cut-off value) was determined, which was completed by Medcalc software. The specific results are as Figure 4 shown. The results showed that the area under the curve AUC of cytoplasmic acyl-CoA thioester hydrolase was 0.83 (the closer the AUC is to 1, the better the diagnostic value. Generally, AUC > 0.5 is considered to have diagnostic value). The optimal diagnostic Cut-off value was 62.5 pg / mL, with a diagnostic sensitivity of 80%, a specificity of 74%, and an accuracy of 77%.

[0031] Example 5 Evaluate the diagnostic value of cytoplasmic acyl-CoA thioester hydrolase as a biomarker in the preparation of a kit for the auxiliary differentiation of AD and VD: The results are as Figure 5 shown. The ROC curve analysis of the expression levels of cytoplasmic acyl-CoA thioester hydrolase in AD and VD patients showed that the area under the curve AUC was 0.89, proving that cytoplasmic acyl-CoA thioester hydrolase can effectively distinguish AD and VD.

[0032] Example 6 Evaluate the correlation between cytoplasmic acyl-CoA thioester hydrolase and Aβ 40 content: Logistic regression analysis was performed using SPSS 14.0 software to evaluate the correlation between the serum level of cytoplasmic acyl-CoA thioester hydrolase and Aβ 40 content, and to judge whether the expression level of cytoplasmic acyl-CoA thioester hydrolase is related to the metabolism of Aβ 40 . The specific results are as Figure 6 shown. The results showed that there was a significant positive correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and Aβ 40 content (r = 0.79; p < 0.001). It is suggested that cytoplasmic acyl-CoA thioester hydrolase may be involved in Aβ 40metabolism

[0033] Example 7 Evaluate the comprehensive diagnostic performance of the combined use of cytoplasmic acyl-CoA thioester hydrolase and ALG-2-interacting protein X in the preparation of an AD auxiliary diagnostic kit: Through ROC curve analysis, evaluate the sensitivity, specificity and accuracy of the combined use of cytoplasmic acyl-CoA thioester hydrolase and ALG-2-interacting protein X in the preparation of an AD diagnostic kit, which is completed by Graphpad Prism software. The specific results are as Figure 7 shown. The results show that the AUC is 0.95 (the closer the AUC is to 1, the better the diagnostic value is considered), the diagnostic sensitivity is 93%, the specificity is 91%, and the accuracy is 92%.

[0034] In summary, the present invention provides effective molecular markers for the differential diagnosis of AD patients and VD patients. The present invention proves through a large number of experiments that cytoplasmic acyl-CoA thioester hydrolase is highly expressed in AD patients. As an AD serum molecular marker, it has a high diagnostic value. When its serum content is higher than 62.5 pg / ml, it can assist in the diagnosis of AD and can also be used as an auxiliary index for distinguishing AD and VD. It is applied to the preparation of a kit for differentiating and diagnosing AD and VD, and has sensitivity, specificity and accuracy, meeting the needs of the staff.

[0035] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the principles described in the specification are only the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker, characterized in that, The biomarker is cytoplasmic acyl-CoA thioester hydrolase.

2. A method for preparing a biomarker, characterized in that, It includes: Extract mRNA from specific biological tissues, such as the brain, reverse transcribe to obtain cDNA, use primers to amplify the cDNA sequence encoding cytoplasmic acyl-CoA thioester hydrolase, and clone the amplified cDNA sequence into an expression vector; Use prokaryotic expression systems such as Escherichia coli or eukaryotic expression systems such as mammalian cells to express the cloned gene; Optimize the protein expression level through induced expression and under appropriate culture conditions; Use appropriate purification techniques to isolate and purify the expressed protein, and the biomarker can be obtained.

3. The preparation method of a biomarker according to claim 2, characterized in that, The induced expression is to use IPTG to induce the expression in Escherichia coli.

4. The preparation method of a biomarker according to claim 2, characterized in that, The purification technique includes any one of affinity chromatography and ion exchange chromatography.

5. Use of a biomarker, characterized in that, It includes: The concentration of cytoplasmic acyl-CoA thioester hydrolase in the sample was detected by ELISA method, and logistic regression analysis was performed to evaluate the correlation between the serum level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment and Aβ content in AD patients and VD patients, and to determine whether the expression of cytoplasmic acyl-CoA thioester hydrolase is closely related to the AD disease process; 40 Content, and to judge whether the expression of cytoplasmic acyl-CoA thioester hydrolase is closely related to the AD disease process; Through ROC curve analysis, judge the sensitivity, specificity and accuracy of cytoplasmic acyl-CoA thioester hydrolase as a biomarker in AD patients and VD patients; Prepare an auxiliary diagnostic kit for differentiating and diagnosing AD and VD with cytoplasmic acyl-CoA thioester hydrolase.

6. The application of a biomarker according to claim 5, characterized in that, The protein expression level of cytoplasmic acyl-CoA thioester hydrolase shows a significant positive correlation with the Aβ 40 content, indicating that cytoplasmic acyl-CoA thioester hydrolase is involved in the 40 metabolism of Aβ.

7. Use of a biomarker according to claim 5, characterized in that, During Logistic regression analysis, there is a significant negative correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) of AD patients, and there is no significant correlation between the protein expression level of cytoplasmic acyl-CoA thioester hydrolase and the degree of cognitive impairment (MMSE score) of VD patients.

8. The application of a biomarker according to claim 5, characterized in that, In ROC curve analysis, the area under the curve AUC for diagnosing AD with cytoplasmic acyl-CoA thioester hydrolase is 0.83, the optimal diagnostic concentration value is 62.5 pg / mL, its diagnostic sensitivity is 80%, specificity is 74%, and accuracy is 77%.

9. The application of a biomarker according to claim 5, wherein, In ROC curve analysis, the area under the curve AUC for differentiating and diagnosing AD and VD with cytoplasmic acyl-CoA thioester hydrolase is 0.89, proving that cytoplasmic acyl-CoA thioester hydrolase can effectively distinguish AD patients and VD patients.

10. The application of a biomarker according to claim 5, wherein The AUC of the AD diagnostic kit prepared by combining the cytoplasmic acyl-CoA thioester hydrolase and ALG-2-interacting protein X is 0.95, the diagnostic sensitivity is 93%, the specificity is 91%, and the accuracy is 92%.

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