Fluorescent probe for detecting C-reactive protein and preparation method thereof

A fluorescent probe and reactive protein technology, applied in the field of molecular biology, can solve the problems of high cost, difficult to obtain, cumbersome preparation of CRP monoclonal antibody, etc., and achieve the effect of good use effect.

Inactive Publication Date: 2019-06-28
CHANGCHUN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the technical defects of the prior art, and provides a fluorescent probe for detecting C-reactive protein and its preparation method, so as to solve the cumbersome preparation of CRP monoclonal antibody in the prior art, which is not easy to obtain and has high cost. question

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  • Fluorescent probe for detecting C-reactive protein and preparation method thereof
  • Fluorescent probe for detecting C-reactive protein and preparation method thereof
  • Fluorescent probe for detecting C-reactive protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Using CRP Standards to Establish Standard Detection Curves for Fluorescent Probes

[0039] 1. Screening of specific affinity ligands (screening includes non-specific elution and specific elution)

[0040] (1) The first round of non-specific elution

[0041] a. Use 0.1mol / L, pH 8.6 NaHCO for CRP 3 The solution was prepared as a 100 μg / mL solution. Take 1 mL of this solution and coat it on a sterile polystyrene petri dish (60×15 mm), and carefully rotate repeatedly until the surface of the petri dish is completely wet. After completion, place the culture dish in a plastic box covered with wet gauze and incubate overnight at 4°C.

[0042] b. After overnight incubation, pour off the coating solution in the petri dish, and shake vigorously on the filter paper that has been irradiated by ultraviolet light to remove the residual coating solution. After removing the residual liquid, add 2 mL of blocking solution to the Petri dish, and block at 4°C for more than 1 hr.

[00...

Embodiment 2

[0110] A specific affinity ligand of C-reactive protein, the amino acid sequence of the ligand is: S-P-H-N-R-S-N-L-V-Q-E-L.

[0111] The preparation method of the above-mentioned specific affinity ligand comprises the following steps:

[0112] 1) Coat CRP, discard the coating solution and add blocking solution to block after coating overnight, then discard the blocking solution and wash with TBST buffer, then mix and incubate with phage, and then wash the phage that specifically binds to CRP take off,

[0113] 2) Repeat step 1) several times; the phages used for mixed incubation in each time were amplified to a quantity of 2×10 after being eluted the previous time. 11 pfu of phage that specifically binds to CRP;

[0114] 3) For the phage specifically bound to CRP obtained in step 2) for the last time, titer determination was carried out, and the number of plaques was about 10 2 The blue spot was randomly picked on the titer determination plate, and the ssDNA was extracted a...

Embodiment 3

[0122] A specific affinity ligand of C-reactive protein, the amino acid sequence of the ligand is: S-P-H-N-R-S-N-L-V-Q-E-L.

[0123] A method for preparing the above-mentioned specific affinity ligand, comprising the following steps:

[0124] 1) Coat CRP, discard the coating solution and add blocking solution to block after coating overnight, then discard the blocking solution and wash with TBST buffer, then mix and incubate with phage, and then wash the phage that specifically binds to CRP take off,

[0125] 2) Repeat step 1) several times; the phages used for mixed incubation in each time were amplified to a quantity of 2×10 after being eluted the previous time. 11 pfu of phage that specifically binds to CRP;

[0126] 3) For the phage specifically bound to CRP obtained in step 2) for the last time, the titer was measured, and blue spots were randomly picked from the titer measuring plate with 30 to 300 plaques, and the ssDNA was extracted and then sequenced .

[0127] Wh...

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Abstract

The invention provides a fluorescent probe for detecting the C-reactive protein and a preparation method thereof. With the phage display technology, the recombinant phage bound specifically is screened; and sequencing and sequence comparison are carried out by extracting the ssDNA of the recombinant to obtain a CRP-specific affinity ligand sequence. The ligand is synthesized based on the solid phase polypeptide synthesis technology and fluorescent labeling is carried out on the ligand to obtain a fluorescent probe that is specifically bound to the CRP. A fluorescent enzyme label plate is coated with a to-be-detected serum sample and a fluorescent probe with the certain concentration is added, wherein the probe can be bound to the CRP specifically; a fluorescent probe that is not bound is washed by using a proper buffer solution and a fluorescence microplate reader detects the fluorescence intensity, wherein the current fluorescence intensity is controlled by the CRP content in the to-be-detected sample strictly. According to the invention, a novel tool is provided for specific recognition and content detection of CRP. The fluorescent probe no only can qualitatively identify the CRPbut also can be used for quantitative detection.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and further relates to a fluorescent probe construction technology, in particular to a fluorescent probe for detecting C-reactive protein and a preparation method thereof. Background technique [0002] C-reactive protein (C-Reactive Protein, CRP), also known as immune defense binding protein, is synthesized in liver cells under the stimulation of interleukin (IL-6), tumor necrosis factor α and other cytokines. Peripheral blood lymphocytes and local macrophages in inflammatory response can also produce a small amount of CRP. CRP cannot pass through the placenta, and can be detected in pleural effusion, ascites, pericardial fluid, and joint fluid except blood, and is widely distributed in the human body. The synthesis rate of CRP in normal human body is 1-10 mg / d, and the synthesis amount is more than 1000 mg / d when acute inflammatory reaction occurs. [0003] Studies have shown that CR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C07K7/08
Inventor 张淑华刘娜王晴罗杰张馨予李颂
Owner CHANGCHUN UNIV OF SCI & TECH
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