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Core amino acid sequence for targeted recognition of gentamicin single-chain antibody and application thereof

A gentamicin, targeted recognition technology, applied in the direction of material inspection products, instruments, analytical materials, etc., can solve the problems of not adapting to the fast and simple market monitoring requirements, high personnel quality requirements, and long testing time. The detection cost is low, time-saving and labor-saving, and the effect of high affinity

Inactive Publication Date: 2019-12-13
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are sensitive and accurate, they are time-consuming, costly, and require high personnel quality, so they are not suitable for fast and simple market monitoring requirements.

Method used

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  • Core amino acid sequence for targeted recognition of gentamicin single-chain antibody and application thereof
  • Core amino acid sequence for targeted recognition of gentamicin single-chain antibody and application thereof
  • Core amino acid sequence for targeted recognition of gentamicin single-chain antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Using the pCANTAB5e system to display recombinant antibody library construction and panning

[0026] 1. Extraction of total RNA from splenocytes

[0027] 1. The mice (unimmunized BALB / c mice) were killed by cervical dislocation, and the mice were soaked in 75% (V / V) alcohol for 10 minutes. Put it into the ultra-clean workbench, open the abdomen with sterilized scissors, and take out the spleen with tweezers. Put the spleen into a plate containing 10 mL of sterilized 0.02M PBS buffer, peel off the surrounding fat tissue, wash the outer surface, and then put it into a plate containing 10 mL of sterilized 0.02M PBS buffer for rinsing once. Thoroughly wash away the free fat cells on the surface of the spleen.

[0028] 2. Place the spleen on a sterilized nylon mesh, cut it into pieces, wash it with 10mL of sterilized 0.15M PBS buffer solution, and grind it with scissors while washing. Add 10mL of sterilized 0.15M PBS buffer according to the remaining amount of ...

Embodiment 2

[0044] Example 2. ELISA Identification of Sequence and Gentamicin Potency and Inhibition

[0045] (1) ELISA identification of the potency of gentamicin

[0046] 1. The gentamicin antigen was coated with 1 μg / mL (protein amount) on the microplate; the PBS buffer was used as the control. Among them, the coated antigen was diluted with carbonate (CBS) buffer, 50 μL per well was added to a 96-well microtiter plate, placed at 4°C overnight, washed 5 times with phosphate Tween PBST buffer, and then Block with 1% bovine serum albumin (BSA) solution.

[0047] 2. Dilute the artificially expressed and purified gentamycin single-chain antibody with PBS buffer (pH 7.4), add it to the above microtiter plate at a volume of 50 μL per well, mix well, and place at 37°C Under conditions, incubate for 30 min in the dark.

[0048] 3. Wash 5 times with PBST buffer, and dry the liquid in the wells of the microtiter plate; use PBST buffer to dilute the avidin coupled with horseradish peroxidase 1...

Embodiment 3

[0061] Embodiment 3. The application of gentamicin single-chain antibody

[0062] Enzyme-linked immunosorbent assay (ELISA) has the characteristics of sensitivity, specificity, simplicity, speed, stability, and ease of automatic operation. If it can be applied to the detection of gentamicin residues, it will greatly reduce the cost of detection and shorten the detection time. Existing experiments have used the EDC method to prepare the complete antigen of gentamicin, immunized mice with the prepared antigen, successfully obtained a hybridoma cell line that stably secretes anti-gentamicin monoclonal antibody after cell fusion and cloning, and has a high The antibody titer is high, the antibody subtype is IgG1, and the antibody has high affinity, which can be used for the detection of aminoglycoside drug residues. The results of the ELISA binding test show that the gentamicin single-chain antibody can have a good binding ability to the corresponding target antigen, thus proving ...

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Abstract

The invention relates to a core amino acid sequence for targeted recognition of a gentamicin single-chain antibody and an application thereof, which belong to the field of antibacterial drug antigen detection. According to the invention, molecular docking and virtual screening technologies are used, on the basis of a gentamicin single-chain antibody structure, a polypeptide ligand with the best binding mode and affinity with a target protein in a virtual polypeptide library is searched through a molecular docking technology, and finally a polypeptide sequence specifically binding to a gentamicin antigen is obtained, and the polypeptide sequences are FCTRRDGYYAV and TTSTDIDDDMN. Through affinity identification with gentamicin antigen, the affinity is good. The ELISA binding test results show that the gentamycin single-chain antibody can be well combined with a corresponding target antigen, and the polypeptide designed by the invention can be used for quantitative and qualitative rapid detection of the gentamycin antigen.

Description

technical field [0001] The invention relates to the amino acid sequence and application of a gentamicin single-chain antibody, belonging to the field of antigen detection of antibacterial drugs. Background technique [0002] Using phage display technology to screen specific single-chain antibodies of aminoglycoside antibacterial drugs, antibodies can be obtained without relying on cell fusion and animal immunization, and the inhibitory effect can be identified by ELISA after expression and purification, and the test period is short. By linking with the NCBI DNA database on the Internet, SWISS-MODEL software was used to model the homology of the scFv gene sequence and analyze its structure. [0003] The virtual screening technology based on molecular docking is an emerging technical means to study the interaction between peptides and proteins. This technology mainly uses fast computer calculations to realize the docking of peptides and corresponding target proteins in spatia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06G01N33/68
CPCC07K7/06G01N33/9446G01N33/68G01N2410/00
Inventor 王方雨张改平牛艳郝慧芳胡曼张颍硕张运尚邓瑞广
Owner HENAN ACAD OF AGRI SCI
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