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

Aflatoxin nano antibody gene pool, construction method and application of aflatoxin nano antibody gene pool as well as aflatoxin B1 nano antibody 2014AFB-G15

A 2014AFB-G15, aflatoxin technology, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., to achieve good stability, reduce production costs, and reduce interference effects

Active Publication Date: 2014-06-18
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
View PDF3 Cites 69 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on nanobodies against aflatoxin B1

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of aflatoxin nanobody gene library

[0038] 1. Animal Immunization

[0039] Buy a 2-year-old male alpaca, immune to aflatoxin B1 antigen (AFB 1 -BSA, Sigma Corporation). After emulsifying 200 μg of aflatoxin B1 antigen with Freund's incomplete adjuvant, the alpaca was injected subcutaneously at multiple points. Immunize once every 2 weeks, 7-10 days after each immunization, blood was collected from the alpaca, and the serum titer was determined by indirect ELISA method. After the first immunization with the highest titer, 10 mL of blood was taken to extract total RNA.

[0040] 2. Construction of cDNA library

[0041] (1) Extraction of total RNA: select the first immunization with the highest titer of alpaca serum, and 7-10 days after immunization, take 10 mL of blood from the alpaca vein, and extract total RNA: use the LeukoLOCK Total RNA Isolation Kit of Life Technology Company to extract Total RNA in alpaca blood.

[0042] (2) cDNA synt...

Embodiment 2

[0074] Example 2 Screening and sequence determination of aflatoxin B1 nanobody

[0075] 1. Panning of Aflatoxin B1 Nanobody

[0076] use AFB 1 -BSA (1μg / well) and 3% BSA-PBS solution (used as a negative control) were coated on the ELISA plate, overnight at 4°C; the next day, after pouring off the coating solution, wash the plate 3 times with PBST, and then wash the plate with 3% Skimmed milk powder was blocked for 1 h; the plate was washed 3 times with PBST, and the plate was coated with AFB 1 -Add 50 μl of the above-mentioned rescued aflatoxin nanobody gene library to the wells of BSA, and incubate at 37°C for 1 h; after washing the plate 10 times with PBST, add 100 μl, 100ng / mL AFB to each well 1Solution, shake at room temperature (20°C~30°C) for 30min to elute. Transfer the eluate to wells coated with 3% BSA-PBS solution, and incubate at 37°C for 1 h (to remove non-specific adsorption); after incubation, take the supernatant and infect 2 mL of TG1 bacterial solution ...

Embodiment 3

[0083] Example 3 Preparation of aflatoxin B1 nanobody 2014AFB-G15

[0084] (1) Obtain the TG1 bacterial liquid capable of secreting aflatoxin B1 nanobody 2014AFB-G15, use Qiagen’s DNA mini-extraction kit to extract the plasmid, transform it into HB2151 competent cells, and spread it on the LB ampicillin plate;

[0085] (2) Pick the HB2151 colony containing the aflatoxin B1 nanobody 2014AFB-G15 plasmid and culture it in 100mL SB ampicillin liquid medium at 250 rpm at 37°C until OD600=0.5-0.8, and add 200μl 0.5M IPTG solution to induce overnight.

[0086] (3) Refrigerate and centrifuge at 10,000 rpm for 15 min at 4°C, carefully remove the supernatant in a sterile operating bench, and extract the soluble protein by osmotic shock to obtain the supernatant protein. After the supernatant was passed through a 0.22 μm filter membrane, it was dialyzed overnight against an equilibration buffer (50 mM phosphate, 300 mM sodium chloride, 20 mM imidazole; pH 7.4).

[0087] (4) Purify t...

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
recovery rateaaaaaaaaaa
reaction rate constantaaaaaaaaaa
Login to View More

Abstract

The invention relates to an aflatoxin nano antibody gene pool, a construction method and application of the aflatoxin nano antibody gene pool as well as an aflatoxin B1 nano antibody 2014AFB-G15. The aflatoxin nano antibody gene pool is prepared by extracting RNA (ribonucleic acid) in alpaca blood after immunization of an aflatoxin B1 antigen, performing specific amplification on a variable region gene of an alpaca heavy chain antibody by adopting an RT-PCR (reverse transcription-polymerase chain reaction) method to obtain an aflatoxin nano antibody VHH gene, and then performing transformation after connecting with a pCANTAB5E (his) vector. The aflatoxin B1 nano antibody 2014AFB-G15 obtained by screening, disclosed by the invention, has the characteristics of organic reagent resistance, high temperature resistance and the like, and is good in stability; the IC50 (half maximal inhibitory concentration) of the aflatoxin B1 nano antibody 2014AFB-G15 to the aflatoxin B1 is 0.66ng / mL, and the cross reactivity of the aflatoxin B1 nano antibody 2014AFB-G15 to the aflatoxins B2, G1,G2 and M1 is 22.6%, 0.95%, 32.1% and 26% respectively.

Description

technical field [0001] The invention relates to aflatoxin nanobody gene library, construction method, application and aflatoxin B1 nanobody 2014AFB-G15. Background technique [0002] Aflatoxins are mainly secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus, and are natural toxic compounds that can cause various damages to humans and animals. More than 20 kinds of aflatoxins have been found so far, among which aflatoxin B1 is the most toxic, its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. As early as 1993, aflatoxin B1 was designated as one of the strongest known carcinogenic chemicals by the Cancer Research Institute of the World Health Organization, that is, Class I carcinogens. my country is an area with heavy aflatoxin pollution, and aflatoxin pollution is likely to exist in various foods and agricultural products, especially corn, peanuts and their products. Therefore, it is of great significance to strength...

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): C40B40/08C40B50/06C12N15/13C12N15/63C07K16/14G01N33/53
CPCC07K16/14C07K2317/22C07K2317/569C07K2317/92C07K2317/94
Inventor 李培武王妍入张奇张兆威丁小霞
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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