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A single-chain antibody that directly recognizes 3-amino-5-morpholinomethyl-2-oxazolidinone and its preparation method and application

A technology of single-chain antibody and morpholinomethyl, which is applied in the field of food safety detection, can solve the problems of complicated and time-consuming operation, inconvenient application, and low recovery rate, and achieve good application prospects, good antigen binding activity, and cost reduction effects

Active Publication Date: 2021-07-09
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the derivatization strategy can realize the immunodetection of AMOZ, it needs to be derivatized before the detection, which is complicated, time-consuming, costly, and usually the recovery rate is low, which is very inconvenient to apply

Method used

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  • A single-chain antibody that directly recognizes 3-amino-5-morpholinomethyl-2-oxazolidinone and its preparation method and application
  • A single-chain antibody that directly recognizes 3-amino-5-morpholinomethyl-2-oxazolidinone and its preparation method and application
  • A single-chain antibody that directly recognizes 3-amino-5-morpholinomethyl-2-oxazolidinone and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1sc

[0047] Construction Screening and Identification of Example 1 scFv Recombinant Plasmid

[0048] 1. Extraction of total mRNA from hybridoma cells

[0049] S1. Consumables and utensils such as PCR tubes and centrifugal tip tweezers related to the RNA experiment operation are completely soaked in 0.1% DEPC solution overnight. Pour the DEPC solution as much as possible the next day, and autoclave it for later use;

[0050] S2. Will culture well about 1×10 7 Hybridoma cells (Liu et al. Food and Agricultural Immunology, 2017, 1-14, doi: 10.1080 / 09540105.2017.1376038) were suspended and added to a 15mL centrifuge tube, centrifuged at 1000r / min to pellet the cells, the supernatant was discarded, and the Absorb as much of the residual liquid as possible;

[0051] S3. Add 2mL Trizol, repeatedly suck with the tip of the pipette, mix the cells, and place at room temperature for 5min;

[0052] S4. Divide 1 mL of this solution into two 1.5 mL RNAase-free centrifuge tubes, add 200 L of c...

Embodiment 2

[0108] Example 2 Extraction of soluble expression of scFv antibody

[0109] The pComb3XSS phagemid carrier that the present invention adopts is the plasmid carrier that contains bacteriophage intergenic region, and it has integrated the advantage of phage and plasmid carrier, and phagemid has an amberstop acid terminator (AmberStop Codon) between multi-cloning site and gm ), when the recombinant phage pComb3XSS-Fab transforms the suppressor Escherichia coli XL1-Blue, the acid terminator is read through into glutamic acid, and as a result, the antibody fusion protein will be expressed on the surface of the phage, that is, the surface display of the phage. When pComb3XSS-Fab is transformed into non-suppressor type E. coli TOP 10F', the acid terminator is recognized as a stop codon, induced by the lactose analog PTG, the antibody is expressed and secreted into the cytoplasm, becoming a soluble molecule.

[0110] Its expression and extraction steps are as follows:

[0111] S1. Pi...

Embodiment 3sc

[0118] Example 3 detection of scFv soluble protein ci-ELISA

[0119] The ci-ELISA detection steps are as follows:

[0120] S1. Coating: Dilute the coating material to an appropriate lug / mL with the coating solution, add 100 μL / well to the wells of the microtiter plate, and place in a 37°C water bath overnight.

[0121] S2. Washing: Pour off the liquid in the wells, wash the plate twice with a plate washer, add 250 μL of washing solution to each well, and dry the liquid in the wells.

[0122] S3. Blocking: add 120 μL of blocking solution to each well, block at 37°C for 3 hours, shake off the liquid in the well, and place it upside down in an oven at 37°C for 1 hour for later use.

[0123] S4. Adding samples and incubating: Dilute AMOZ into a series of gradient standard solutions, dilute to 8000, 2000, 500, 125, 31.25, 7.8125, 1.95313, 0.48828ng / mL, add 50 μL to each well, and then add a reasonable dilution of scFv protein to dilute 50 μL of washing solution was diluted and sh...

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PUM

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Abstract

The invention discloses a single-chain antibody (scFv) directly recognizing 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), its preparation method and application. The single-chain antibody includes a heavy chain variable region and a light chain variable region, the sequences of which are shown in SEQ ID NO. The sequences shown have the same functional amino acid sequence. The binding of the single-chain antibody scFv provided by the invention to the antigen can be competitively inhibited by the free hapten, IC 50 The detection limit is 72.93ng / mL, and the detection limit is 13.35ng / mL, which has good antigen binding activity; the linear detection range is between 24.98~212.92ng / mL through the standard curve. The genetically engineered antibody has good application prospects in the immunodetection of AMOZ and the rapid detection and screening of a large number of samples.

Description

technical field [0001] The invention belongs to the technical field of food safety detection. More specifically, it relates to a single-chain antibody (scFv) directly recognizing 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), its preparation method and application. Background technique [0002] 3-Amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) is an in vivo metabolite of the nitrofuran antibiotic furaltadone. After furaltadone is taken by animals, the original drug molecule is rapidly metabolized in the animal body, and its stability in the body does not exceed a few hours, but its metabolite AMOZ binds to the cell membrane protein and becomes a bound state, which can remain stable in the body for a long time, thereby delaying The clearance rate of the original drug in the body. However, ordinary food processing methods (such as barbecue, microwave processing, cooking, etc.) are difficult to degrade a large amount of bound AMOZ residues, and eating meat products contai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N15/70G01N33/58
CPCC07K16/44C07K2317/56C07K2317/622C12N15/70G01N33/581G01N2430/00
Inventor 徐振林陈子键王弘沈玉栋肖治理雷红涛杨金易孙远明
Owner SOUTH CHINA AGRI UNIV
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