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A kind of short peptide, detection kit and method for detecting vascular endothelial growth factor

A detection kit and short peptide technology, applied in the fields of biotechnology and immunoassay, can solve the problems of cumbersome operation process, low degree of automation and cumbersome preparation process of the detection method, and achieve low cost, low commercialization cost and good safety. Effect

Active Publication Date: 2022-03-15
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors of the present invention found that although antibodies and aptamers have good selectivity as capture probes, their inherent weaknesses (such as cumbersome preparation process, low stability and high cost) limit their application, In addition, the existing detection methods are not only cumbersome to operate, but also have a low degree of automation and are greatly affected by human factors.

Method used

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  • A kind of short peptide, detection kit and method for detecting vascular endothelial growth factor
  • A kind of short peptide, detection kit and method for detecting vascular endothelial growth factor
  • A kind of short peptide, detection kit and method for detecting vascular endothelial growth factor

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Embodiment approach

[0026] A typical embodiment of the present invention provides a short peptide, which is polypeptide 1 or polypeptide 2;

[0027] The polypeptide 1 is: a polypeptide consisting of an amino acid sequence of LSTSPHTGGGSC;

[0028] The polypeptide 2 is: a polypeptide derived from the polypeptide 1 through substitution and / or deletion and / or addition of one or several amino acid residues and having the same function.

[0029] It was found through experiments that the short peptide not only has the advantages of high affinity and high selectivity for VEGF165, but also has the advantages of small molecular weight and good stability. The short peptide is a short-chain polypeptide. The short peptide is moderately extended while ensuring the binding site, so as to enhance the binding efficiency with the polystyrene surface, and at the same time facilitate more sufficient exposure of the probe binding site.

[0030] Another embodiment of the present invention provides a detection kit, ...

Embodiment 1

[0045] Probe V for specificity testing:

[0046] Dissolve the specific synthetic probe V in 0.1M TBS (pH 7.4) to a concentration of 10 μg / mL, then load 100 μL of the prepared solution into the wells of a 96-well microtiter plate, and store the sealed plate at 4 °C Incubate overnight; the next day, suck out the synthetic peptide probe solution in the ELISA plate, place the plate face down on a clean paper towel, pat dry the residue, add 0.5% TBST to rinse: 200 μL, 6 times, 2 min each time; antigen ( 5ng / mL, 100μL): vascular endothelial growth factor 165 (VEGF165), vascular endothelial growth factor 121 (VEGF121), human immunoglobulin G (HIgG), human immunoglobulin A (HIgA), human serum albumin (HSA) , prostate specific antigen (PSA), carbohydrate antigen 15-3 (CA15-3), cancer antigen 19-9 (CA19-9), carbohydrate antigen 242 (CA242) and carcinoembryonic antigen (CEA) were placed in 96 After incubating the well plate at room temperature for 3 hours, wash 6 times with TBST and pat...

Embodiment 2

[0049] Probe V optimal coating concentration test:

[0050] Serial serial dilutions of specific probe V: 2, 5, 10, 20, 30, 40 μg / mL were added to the wells of a 96-well white microplate, 100 μL per well, wrapped with plastic wrap, and placed in 4 Incubate at ℃ overnight with shaking; suck out the synthetic peptide probe solution in the ELISA plate, place the plate face down on a clean paper towel, pat dry the residue, add 0.5% TBST to rinse: 200 μL per well, wash 6 times, 2 min each time ; Add 1 μg / mL VEGF165 100 μL to the well one by one, incubate with shaking at room temperature for 3 h; wash 6 times with TBST and pat dry; dilute the anti-VEGF165 polyclonal antibody in the buffer to a concentration of 1:1000, add 100 μL to the well And shake and incubate at room temperature for 1 hour, then wash 6 times with TBST and pat dry; Dilute the IgG-HRP monoclonal antibody in the buffer to a concentration of 1:500, add 100 μL of the diluted conjugate to each well, and incubated at r...

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Abstract

The invention discloses a short peptide, a detection kit and a method for detecting vascular endothelial growth factor. The short peptide is polypeptide 1 or polypeptide 2; the polypeptide 1 is a polypeptide composed of an amino acid sequence of LSTSPHTGGGSC; the polypeptide 2 is : a polypeptide derived from polypeptide 1 through substitution and / or deletion and / or addition of one or several amino acid residues and having the same function. The short peptide is used as a capture antibody, combined with a chemiluminescent enzyme immunoassay technique (CLIA) to detect a designated tumor marker in serum. The solid-phase biological polypeptide probe is used to incubate the target protein in the serum sample to be detected, and then it can be effectively detected under the catalytic chromogenic system. The method provided by the invention has strong specificity, high sensitivity and convenient operation, and at the same time, the cost of making and purifying the probe is relatively low, and provides an effective way for realizing sensitive and rapid detection of VEGF165.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and immune analysis, and relates to a short peptide, a detection kit and a method for detecting vascular endothelial growth factor. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Vascular endothelial growth factor (VEGF) is a highly glycosylated basic protein with an isoelectric point of 8.5 and a molecular weight of 45KD. It is formed by combining two identical subunits through disulfide bonds and has strong acid and heat resistance. VEGF is an extremely important cytokine that regulates neovascularization. There are five different subtypes of VEGF, namely VEGF165, VEGF189, VEGF206, VE...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08G01N33/68G01N33/577
CPCC07K7/08G01N33/68G01N33/577G01N2333/515G01N2410/00
Inventor 刘爱骅王戈
Owner QINGDAO UNIV
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