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Aflatoxin M1 anti-idiotype nano antibody and preparation method thereof

A technology of aflatoxin and nano-antibody, which is applied in the field of molecular biology, can solve the problems of harmfulness to the operator and the environment, and achieve the effects of reducing production costs, good diversity, and high storage capacity

Active Publication Date: 2021-07-23
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, pure toxins used as calibrators are hazardous to both operators and the environment

Method used

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  • Aflatoxin M1 anti-idiotype nano antibody and preparation method thereof
  • Aflatoxin M1 anti-idiotype nano antibody and preparation method thereof
  • Aflatoxin M1 anti-idiotype nano antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of Phage Display Nanobody Immune Library

[0028] 1. Alpaca immunity

[0029] anti-aflatoxin M 1 Monoclonal antibody (dissolved in PBS7.4) 200 μg, emulsified with an equal volume of Freund's incomplete adjuvant, subcutaneously injected into three-year-old male alpaca (Alpaca), and then immunized once every two weeks. 8 times, blood testing began after the fourth immunization. Seven to 10 days after the fourth immunization, 10 mL of blood was collected from the jugular vein of an EDTA vacuum blood collection tube. At the same time, the blood collection tube was gently inverted to avoid blood coagulation, and then the blood was processed with the filter in the LeukoLOCK kit, and then sequentially washed with 3 mL of LPBS buffer and 3 mL of RNAlater Wash the filter with the buffer solution, and the required white blood cells will be trapped in the filter, and finally the filter should be sealed and stored at -80°C for RNA extraction.

[0030] 2. T...

Embodiment 2

[0056] Embodiment 2 Aflatoxin M 1 Panning and identification of anti-idiotypic nanobodies

[0057] (1) Aflatoxin M 1 Panning of anti-idiotypic nanobodies

[0058] aflatoxin M 1 Monoclonal antibody is the target (the aflatoxin M 1 The monoclonal antibody is secreted by the hybridoma cell line 2C9 with the preservation number CCTCC NO.C201018. The hybridoma cell line 2C9 has been preserved in the China Center for Type Culture Collection (CCTCC) on July 13, 2010, and has been listed in the patent "Hybridoma cell line 2C9, its anti-aflatoxin M 1 Monoclonal Antibody and Its Application", application number: 201110108230.6), the aflatoxin M that was eluted by reducing the original concentration of the coating and competing 1 Concentration of standard substance, alternately use blocking reagents, carry out affinity enrichment panning, and obtain aflatoxin M 1 Monoclonal antibody antibodies, namely anti-antibodies, also known as anti-idiotypic antibodies, panning steps are as fo...

Embodiment 3

[0083] Embodiment 3 Aflatoxin M 1 Expression and purification of anti-idiotypic nanobodies

[0084] (1) Preparation of Top10F' competent cells:

[0085] (1) Pick an appropriate amount of Top10F' to streak on the LB-tetracycline plate, and culture overnight at 37°C;

[0086] (2) Pick the Top10F' monoclonal into 5mL LB medium, culture at 37°C, 250rpm until OD 600 0.6-0.8;

[0087] (3) Place the bacterial solution on ice for 30 minutes;

[0088] (4) Centrifuge at 5000g for 5min at 4°C, put it back on ice quickly, discard the supernatant, and add 1mL of pre-cooled 0.1M CaCl 2 Resuspend the bacteria in the solution, pipette and mix well, and then ice-bath for 7 minutes;

[0089] (5) Repeat the above operation once;

[0090] (6) Centrifuge at 5000g for 5min at 4°C and quickly put it back on ice, completely absorb the liquid, and use 100μL pre-cooled 0.1MCaCl for precipitation 2 The solution is resuspended, which is Top10F'competent cells.

[0091] (2) Extraction and transfor...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to an aflatoxin M1 anti-idiotype nano antibody and a preparation method thereof. An amino acid sequence of the aflatoxin M1 anti-idiotype nano antibody is as shown in SEQ ID NO:1. According to the invention, alpaca is immunized by an aflatoxin M1 monoclonal antibody, a phage display nano antibody library is constructed by extracting total RNA of leukocytes of the alpaca, and the library is high in capacity and good in diversity; the aflatoxin M1 monoclonal antibody is used as a target object, four rounds of affinity enrichment panning are adopted, the coating concentration and the competitive elution concentration are reduced round by round, and a positive bacteriophage aflatoxin M1 anti-idiotype nano antibody with good specificity is obtained through panning; and a positive bacteriophage aflatoxin M1 anti-idiotype nano antibody gene is transformed into an expression strain, and a protein-form nano antibody VHH C4 with excellent performance is obtained through induced expression, purification and identification.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a kind of aflatoxin M 1 Anti-idiotypic nanobodies and methods for their preparation. Background technique [0002] Aflatoxins are metabolites produced by Aspergillus flavus or Aspergillus parasiticus, which contaminate a variety of agricultural products and are found during crop growth, harvesting, storage and processing. These metabolites are highly toxic, mutagenic, teratogenic and carcinogenic compounds that have been implicated as causative agents of human hepatic and extrahepatic carcinogenesis. Aflatoxin B 1 (AFB 1 ) is the most toxic and is classified as a Group 1 human carcinogen by the International Agency for Research on Cancer. AFM 1 is AFB 1 hydroxylated metabolites. uptake by AFB 1 Lactating animals fed contaminated feed will incorporate AFM 1 It is excreted in milk, so it can be found in a wide variety of milk products. AFM 1 Also toxic and carcinogenic, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C12N15/13C12N15/70
CPCC07K16/4208C07K16/005C12N15/70C07K2317/22C07K2317/569C07K2317/33C07K2317/14
Inventor 唐晓倩李培武王督印南日张奇
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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