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Specific epitope peptide of occludin 31kDa degradation fragment and application of specific epitope peptide

An epitope and specific technology, applied in the biological field, can solve the problem of slow rising speed, and achieve the effect of low detection limit, good specificity and improved safety.

Active Publication Date: 2019-11-19
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2) 2 hours after ischemia, the level of 55kDa fragments can be found to increase in peripheral blood, but the normal baseline level of serum (that is, before cerebral ischemia) is also high, and the rate of increase is slow

Method used

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  • Specific epitope peptide of occludin 31kDa degradation fragment and application of specific epitope peptide
  • Specific epitope peptide of occludin 31kDa degradation fragment and application of specific epitope peptide

Examples

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Effect test

Embodiment 1、31

[0022] Example 1, Design and Synthesis of Antigenic Epitope Peptide of 31kDa Occlusin Degradation Fragment

[0023] Analysis of human Occludin protein (occlusive protein) amino acid sequence (shown in SEQ ID No.1), Occludin protein 31kDa degradation fragment amino acid sequence (321-522 shown in SEQ ID No.1), and Occludin protein 55kDa degradation fragment amino acid Sequence position, select three amino acid sequences in the amino acid sequence of the 31kDa degradation fragment of Occludin protein as candidate specific epitopes of the 31kDa degradation fragment of Occludin protein, and synthesize three polypeptides respectively, namely antigen epitope peptides T1, T2, and T3. The sequences are as follows:

[0024] Antigenic epitope peptide T1: 323-334 positions shown in SEQ ID No.1;

[0025] Antigen epitope peptide T2: No. 346-355 shown in SEQ ID No.1;

[0026] Antigen epitope peptide T3: 363-373 positions shown in SEQ ID No.1.

Embodiment 2

[0027] Embodiment 2, the preparation of antigen and antibody

[0028] The three polypeptides (antigenic epitope peptides) obtained in Example 1 were respectively linked to the carrier protein KLH (hemocyanin) to prepare three immunogenic antigens.

[0029] The three antigens were used as immune antigens respectively, and the rabbits were immunized by subcutaneous injection, and the immunization was boosted once every two weeks, a total of 4 immunizations. Blood was collected from the carotid artery to obtain antiserum, and then purified to obtain the three antibodies.

[0030] Using the ELISA method, the corresponding antigenic epitope peptide-BSA was used as the detection antigen, and the titers of the antisera containing the corresponding antibodies were tested respectively, and the results were all above 1:3000.

Embodiment 3

[0031] Embodiment 3, Western blot detection antibody specificity

[0032] Take a sample of peripheral blood serum from 3 patients with acute ischemic stroke, perform protein electrophoresis separation and membrane transfer, and then use the three antibodies prepared in Example 2 to detect each sample separately, and use Occludin protein to detect The antibody was used as a control, and the results are shown in Table 1.

[0033] Table 1. Antibody specificity results detected by Western blot

[0034]

[0035] Note: "-" in Table 1 indicates that the result is negative, and "+" indicates that the result is positive.

[0036] The results in Table 1 show that the antibody KT-1 has the specificity of recognizing the 31kDa degraded fragment of atretin but cannot recognize the 55kDa degraded fragment of atretin and the undegraded atretin protein. Antibody KT-2 and antibody KT-3 do not have the specificity of recognizing the 31kDa degraded fragment of atretin specificity.

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Abstract

The invention discloses a specific epitope peptide of an occludin 31kDa degradation fragment and application of the specific epitope peptide. The specific epitope peptide comprises an amino acid sequence shown in the site 323-334 of SEQ ID No.1. An antibody generated by an antigen prepared from the specific epitope peptide is only capable of recognizing the occludin 31kDa degradation fragment, butcannot recognize an occludin 55kDa degradation fragment or undegraded occluding, and has the advantages of being good in specificity and low in detection limit. By adopting the specific epitope peptide, a corresponding novel detection technique is provided for rapidly and efficiently testing the level of a 31-kDa occludin fragment in serum of a patient suffering from acute ischemic stroke at a super early stage, and predicting cerebral hemorrhage in recanalization treatment on acute ischemic stroke.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific antigenic epitope peptide of a 31kDa degradation fragment of occludin and its application. Background technique [0002] Currently, neither CT (Computed Tomography, Computerized Tomography) nor MRI (Magnetic Resonance Imaging, Magnetic Resonance Imaging) can accurately and rapidly measure the damage of the blood-brain barrier before thrombolysis. Several serological markers (including MMP-9 and cFn) that have been placed with high hopes are not only not structural proteins of the blood-brain barrier, but their serum levels are also easily affected by other factors. All in all, it is difficult to accurately reflect the degree of blood-brain barrier damage . [0003] Occludin protein is one of the key molecules that form the "seal" of the blood-brain barrier. A large number of studies have shown that the generation of degraded fragments of occludin indicates the seriousnes...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18G01N33/68G01N33/558
CPCC07K14/47C07K16/18G01N33/558G01N33/6893G01N2333/47G01N2800/2871
Inventor 刘文兰徐迹张圆叶秀峰
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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