Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation and application of nano antibody capable of specifically recognizing fenitrothion

A nanobody, fenitrothion technology, applied in the direction of application, specific peptide, botany equipment and methods, etc., can solve the problem that fenitrothion is not provided

Active Publication Date: 2021-09-14
SOUTH CHINA AGRI UNIV
View PDF7 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the description of the Chinese patent, it can be known that when the patent detects fenitrothion by indirect ELISA method, IC50=13μg / L, which shows that the ultra-high sensitivity detection method for fenitrothion has not been provided yet

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation and application of nano antibody capable of specifically recognizing fenitrothion
  • Preparation and application of nano antibody capable of specifically recognizing fenitrothion
  • Preparation and application of nano antibody capable of specifically recognizing fenitrothion

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] 1. Preparation of immune antigen and detection antigen

[0060] The design and synthesis of the fenitrothion hapten was completed by the preliminary work of the laboratory, and two types of hapten structures were used in this experiment. Hapten structure A is used for the preparation of artificial immunization antigen, and hapten structure B is used for the preparation of artificial detection antigen. The structure of the above-mentioned hapten and artificial antigen is as follows: figure 1 shown.

[0061] Hapten A was coupled with lactoferrin (Lactoferrin, LF) by active ester method to form immune antigen. The specific operation method is as follows: Weigh 7.3mg EDC and 4.3mg NHS, add 0.1mL DMF to dissolve the solids. Add 9.7 mg of fenitrothion hapten A to the above mixture solution, stir or shake at room temperature in the dark for 4 hours to activate the hapten. Weigh 50mg LF, add 5mL carbonate buffer (pH 9.4) to prepare 10mg / mL protein solution. Add the activat...

Embodiment 2

[0097] Example 2 Affinity panning and identification of nanobodies

[0098] 1. Experimental method

[0099] 1. Affinity panning of nanobodies

[0100] (1) Detection of antigen immobilization

[0101] The detection antigen B-BSA was diluted to 1 μg / mL with PBS, added to the microwells of the microplate, 100 μL per well, and left at 4°C overnight. The next day, after washing the plate twice with PBST (0.01M PBS, 0.05% Tween-20), 150 μL of 1% BSA-PBS (w / v) solution was added to each well and allowed to stand at 37° C. for 1 h. Pour off liquid in wells and pat dry on absorbent paper for later use.

[0102] (2) Positive phage screening

[0103] Add BSA to the phage library in Example 1 so that the final concentration of BSA is 1% (w / v), add the mixture to 3 microwells with immobilized antigen, add 100 μL to each well, and incubate at 37° C. for 1 h. The free phage in the wells were discarded, the microwells were washed 10 times with PBST, and then washed 5 times with PBS. Add...

Embodiment 3

[0127] Example 3 Expression of Nanobodies as Single Domain Antibodies

[0128] The Ecoli.TG1 strain carrying the expression vector of the nanobody sm6 (amino acid sequence shown in SEQ ID NO.1) gene after gene sequencing was extracted with a kit, and transformed into competent E.coil BL21 (DE3) And spread on LB-Agar-Amp plate culture to obtain a single colony. Pick a single colony and inoculate it in 10mL LB-Amp, and culture overnight at 37°C and 250rpm. The overnight culture was inoculated into 100mL LB-Amp at a ratio of 1:100, cultured at 37°C, 250rpm to the logarithmic phase, added IPTG to the working concentration, and cultured overnight at 37°C, 250rpm. Centrifuge the bacterial solution and discard the supernatant, redisperse the pellet in 10mL PBS, freeze at -80°C for 30min, and thaw in a water bath at 37°C, repeat this process 3 times, sonicate for 30min, shake at 250rpm for 2h to fully extract the protein, and centrifuge to save the supernatant , the supernatant was ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention discloses preparation and application of a nano antibody capable of specifically recognizing fenitrothion. The amino acid sequence of the nano antibody is as shown in SEQ ID NO. 1; and the nucleotide sequence of a gene for coding the nano antibody is as shown in SEQ ID NO. 9. The nano antibody disclosed by the invention can be applied to actual sample detection of fenitrothion residues, can specifically detect fenitrothion, has the characteristics of relatively high thermal stability, organic tolerance, weak acid resistance and the like, can improve the sensitivity of fenitrothion immunodetection, and is simple to operate and relatively short in consumed time. The method for preparing the nano antibody disclosed by the invention has general applicability, can be used for screening and preparing other small molecular substance nano antibodies, and has very high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of a nanobody specifically recognizing fenitrothion. Background technique [0002] Fenitrothion, also known as fenitrothion, is a widely used organophosphorus pesticide. Fenitrothion has good contact and stomach poisoning effects, and can control pests such as Hemiptera and Coleoptera. It is often used for pest control on corn, rice, soybeans and vegetables. Fenitrothion is moderately toxic. Unreasonable application of fenitrothion will lead to residual accumulation of fenitrothion on crops. Excessive intake has a certain toxic effect on humans and animals: lead to the accumulation of single bonds in the nervous system, causing nausea and vomiting , abdominal pain, diarrhea, coma and other symptoms, acute poisoning can also be life-threatening. [0003] Accurate detection of fenitrothion residues in crops is an important means to avoid da...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N15/13C12N15/70C12N1/21G01N33/58G01N33/543G01N33/53C12R1/19
CPCC07K16/44C07K16/005C12N15/70G01N33/581G01N33/54306G01N33/5308C07K2317/569C07K2317/22C07K2317/565C07K2317/567C07K2317/33
Inventor 徐振林张译丰陈子键王弘罗林沈玉栋雷红涛杨金易肖治理
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com