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Antigenic mimic epitope of gentamycin and application thereof

A gentamicin and mimetic epitope technology, which can be used in material testing products, biological testing, peptides, etc., can solve the problems of increasing testing costs, restricting the application and promotion of immunological methods, and easily causing harm to testing personnel and the environment. To achieve the effect of reducing the harm to human health, saving cost and good effect

Inactive Publication Date: 2014-08-06
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method must use GM standard products, which not only increases the detection cost, but also easily causes harm to the detection personnel and the environment, which restricts the application and promotion of immunological methods to a certain extent.

Method used

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  • Antigenic mimic epitope of gentamycin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Affinity panning and identification of gentamicin antigen mimotope

[0018] 1) Affinity panning of gentamicin antigen mimotope: the specific method is: dilute anti-gentamicin monoclonal antibody with 10 mM PBS (pH 7.4), and coat 96 wells with a final concentration of 100 μg / mL ELISA plates were incubated overnight at 4°C. The next day, after washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 4°C for 2 hours. After 2 hours, discard the blocking solution, wash 5 times with TBST, add 100 μl phage peptide library (phage display ring heptapeptide library or dodecapeptide library, purchased from NEB Company, dilute the phage stock solution 10 times with TBS, about 1.0× 10 11 pfu), shake and react for 1 hour at 22-26°C. Unbound phages were discarded, washed 10 times with TBST, bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2), and immediately neutralized with 15 μl ...

Embodiment 2

[0022] Example 2. Sequencing of Gentamicin Antigen Mimotope Encoding Gene and Determination of its Amino Acid Sequence

[0023] The phages identified by indirect competition ELISA displaying the mimotope of gentamicin antigen were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: carry out phage amplification, after the first step of centrifugation, transfer 800 μl of phage-containing supernatant into a new centrifuge tube. Add 200 μl PEG / NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100 μl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 μl absolute ethanol to precipitate DNA, centrifuge and wash with 70% ethanol Precipitation (DNA sequencing template). The pellet was finally resuspended in 20 μl of sterilized water, and 2 μl was taken for agarose gel electrophoresis analysis; 5 μl of phage template was used for DNA sequencing, and its -96 gIII sequencing primer was: ...

Embodiment 3

[0024] Example 3. Application of Gentamicin Antigen Mimotope as Competing Antigen in ELISA

[0025] (1) Sample extraction

[0026] Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH=7.2)

[0027] After mixing, the sample extract is ready for use.

[0028] (2) Coating and sealing

[0029] Dilute anti-gentamycin monoclonal antibody with 10 mM PBS (pH 7.4), coat the microtiter plate with 10 μg / mL, and incubate overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v / v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, then wash the plate 6 times with PBST stand-by.

[0030] (3) Establishment of standard curve

[0031] Take out the plates treated in step (2), and put 50 μl of phage displaying gentamicin antigen mimotope (1.0×10 1...

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Abstract

The invention belongs to the field of biotechnology and relates to an antigenic mimic epitope of gentamycin, of which the amino acid sequence is WHWQWWLQTDAT. The antigenic mimic epitope of gentamycin can replace an expensive and strong-toxicity gentamycin standard substance and serve as a competitive antigen or a solid coat antigen to be applied to immunological detection of gentamycin. The antigenic mimic epitope of gentamycin has immune reaction characteristics similar to that of natural gentamycin molecules, has a very good effect, reduces harm of gentamycin to the human health, saves the cost and has a very high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mimetic epitope of gentamicin antigen and its application. Background technique [0002] Gentamicin (Gentamicin, GM) is an aminoglycoside antibiotic with ototoxicity and nephrotoxicity, which can cause degeneration and permanent changes in sensory hair cells and damage vestibular nerve function and cochlear nerve. This drug is mainly excreted through the kidneys And accumulate in the kidney, damage the proximal tubular epithelial cells and renal tubular epithelial cells. If people eat animal food with excessive GM for a long time, it will accumulate in the human body and cause serious harm to the human body. [0003] Currently, detection methods for screening illegally added gentamicin include thin-layer chromatography and high-performance liquid chromatography. Thin-layer chromatography is easy to operate, takes a short time, and requires low-cost equipment, but it i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/11G01N33/68
Inventor 何庆华许杨
Owner NANCHANG UNIV
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