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Heptapeptide Antigen Mimic Epitope of Norfloxacin and Its Application

A technology of mimetic epitope and norfloxacin, which is applied in the fields of peptides, material testing products, instruments, etc., can solve the threat to the health of inspectors and the environment, restrict the application and promotion of immunological detection methods, and the price of norfloxacin is expensive and carcinogenic. Sex and other issues, to achieve the effect of good effect, cost saving, and reducing the harm to human health

Inactive Publication Date: 2016-08-03
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of establishing an immunological detection method, standard norfloxacin must be used as a raw material to prepare a competing antigen or a solid-phase coating antigen. It poses a great threat to the health of personnel and the environment, thus restricting the application and promotion of immunological detection methods to a certain extent.

Method used

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  • Heptapeptide Antigen Mimic Epitope of Norfloxacin and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Affinity panning and identification of norfloxacin antigen mimotope

[0024] 1) Affinity panning of norfloxacin antigen mimotope: the specific method is: dilute the anti-norfloxacin monoclonal antibody with 10mMPBS (pH7.4), and coat the 96-well enzyme label with a final concentration of 100μg / mL 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 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. The unbound phages were discarded, washed 10 times with TBST, the bound phages were eluted with 0.2MGlycine-HCl (pH2.2), and immediately neutralized ...

Embodiment 2

[0028] Example 2. The sequencing of the mimotope encoding gene of norfloxacin antigen and the determination of its amino acid sequence

[0029] The phages identified by indirect competition ELISA displaying mimotopes of the norfloxacin 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 (pH8.0), 1 mM EDTA, 4 M NaI), add 250 μl absolute ethanol to precipitate DNA, and wash the pellet (DNA sequencing template) with 70% ethanol after centrifugation. The pellet was finally resuspended in 20 μl sterilized water, and 2 μl was taken for agarose gel electrophoresis analysis; 5 μl of phage template was taken for DNA sequencing, and its -96gIII seq...

Embodiment 3

[0030] Embodiment 3. Norfloxacin antigen mimic epitope is used as the application of competition antigen in ELISA

[0031] (1) Sample extraction

[0032] Weigh 5g of sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200rpm for 5 minutes; filter the extract with Whatman No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS (phosphate buffer, pH=7.2)

[0033] After mixing, the sample extract is ready for use.

[0034] (2) Coating and sealing

[0035] Dilute the anti-norfloxacin monoclonal antibody with 10mMPBS (pH7.4), coat the microtiter plate with 10μg / mL, and incubate overnight at 4°C. The next day, wash with PBST (10mMPBS, 0.05%Tween-20 (v / v)) 3 times, block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, wash the plate 6 times with PBST for use .

[0036] (3) Establishment of standard curve

[0037] Take out the strips treated in step (2), and put 50 μl of phage (1.0×10 11 pfu) and a series of 50 μl...

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Abstract

The invention belongs to the field of biotechnology and relates to 7-peptide mimic antigen epitope of norfloxacin, and the amino acid sequence of the mimic epitope is CNTARWPFC. The 7-peptide mimic antigen epitope of norfloxacin can be used for replacing a norfloxacin standard with high cost and toxicity, serves as a competition antigen or a solid-phase coating antigen to be applied to immunological detection of norfloxacin, has immune response characteristics similar to natural norfloxacin molecules, and has a very good effect. Harm of norfloxacin to human bodies is reduced, the cost is saved, and a very high application value is provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to norfloxacin antigen mimic epitopes and applications thereof. Background technique [0002] Norfloxacin (NOR) is a broad-spectrum antibiotic. It can absorb water in the air, and its color will gradually become darker when exposed to light. It is slightly soluble in dimethylformamide and very slightly soluble in water or ethanol. It is soluble in acetic acid, Soluble in hydrochloric acid or sodium hydroxide solution, it has high antibacterial activity against aerobic gram-negative bacilli, and has good antibacterial effect on most bacteria in vitro, such as most bacteria of Enterobacteriaceae, including Citrobacter, cloacae Enterobacter, Enterobacter aerogenes, Escherichia coli, Klebsiella, Proteus, Salmonella, Shigella, Vibrio, Yersinia, etc. Norfloxacin also has antibacterial activity against multidrug-resistant bacteria in vitro. It also has good antibacterial effect o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K7/08C12N15/11G01N33/68G01N33/577
Inventor 何庆华许杨熊争平
Owner NANCHANG UNIV
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