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Fluorescent probe for detection of cystatin C and construction method thereof

A technology for fluorescent probes and construction methods, which can be used in biological testing, material inspection products, peptides, etc., and can solve problems such as low sensitivity, radioactive contamination, and cumbersome operations

Active Publication Date: 2016-12-07
长春技特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has accurate results, strong specificity, and high sensitivity, but there is radioactive contamination, cumbersome operation, and long detection time
Latex particle method is an immunoturbidimetric assay, which is simple and fast to operate, with small coefficient of variation and error, easy to automate, but low detection sensitivity
Latex-enhanced immunoturbidimetric method is not easily affected by interference factors, has strong specificity, and is convenient for automatic analysis, but the detection cost is high and the sensitivity is low

Method used

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  • Fluorescent probe for detection of cystatin C and construction method thereof
  • Fluorescent probe for detection of cystatin C and construction method thereof
  • Fluorescent probe for detection of cystatin C and construction method thereof

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Embodiment 1

[0084] The establishment of embodiment 1 Cys-C detection method

[0085] 1. Screening of specific affinity ligands (screening includes non-specific elution and specific elution)

[0086](1) The first round of non-specific elution

[0087] a. Use 0.1mol / L, pH 8.6 NaHCO for Cys-C 3 The solution was prepared as a 100 μg / ml solution. Take 1ml of this solution and coat it in a sterile polystyrene petri dish (60×15mm), 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.

[0088] 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 2ml of blocking solution to the Petri dish, and block at 4°C for more than 1 hr.

[0089] c. After blocking, remo...

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Abstract

The invention discloses a fluorescent probe for detection of cystatin C and a construction method thereof, relates to the molecular biological and microbiological technical fields, and is characterized in that the construction method comprises the steps: with Cys-C as a target, screening to obtain a Cys-C specific-binding recombinant phage by using a phage surface random displayed 12-mer peptide library, extracting single stranded DNA of the recombinant phage, carrying out sequencing and sequence comparison, to obtain a sequence Gln-Val-Asn-Gly-Leu-Gly-Glu-Arg-Ser-Gln-Gln-Met of a Cys-C specific affinity ligand having the molecular weight of 1346.5, then carrying out solid phase polypeptide synthesis and fluorescence labeling to obtain the fluorescent probe FITC-Acp-Gln-Val-Asn-Gly-Leu-Gly-Glu-Arg-Ser-Gln-Gln-Met having the molecular weight of 1848.7 and specifically bond with the Cys-C. The fluorescent probe and the construction method thereof have the beneficial effects: 1, a brand new 'tool' is provided for specific identification and content detection of the Cys-C; and 2, the fluorescent ligand peptide probe not only can qualitatively identify the Cys-C, but also can quantitatively detect the content of the Cys-C in samples.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology and microorganisms. Specifically, the invention relates to screening a ligand sequence specifically binding to cystatin C (Cys-C) by using a random display peptide library on the surface of a phage, and synthesizing it with a solid-phase polypeptide. The ligand was synthesized by method, and it was fluorescently labeled to prepare a fluorescent probe for detecting cystatin C. Background technique: [0002] Markers such as urine protein and serum creatinine can be used to detect kidney lesions, but early detection cannot be achieved. Therefore, searching for new detection markers and establishing new detection methods have always been the hotspots of people's research. Since Cys-C was first isolated and purified from egg white by researchers in 1983, a large number of studies have shown that Cys-C has become an irreplaceable effective marker in the early detection of kidney lesions. ...

Claims

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Application Information

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IPC IPC(8): C07K7/08G01N33/68
CPCC07K7/08G01N33/68
Inventor 张淑华陈玉娟及雪敏李成玉杨羽齐凡
Owner 长春技特生物技术有限公司
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