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Cry1ab toxin mimetic antigen based on anti-idiotypic nanobody and its application

A nanobody, anti-idiotype technology, applied in the fields of genetic engineering antibodies and food biology, can solve the problems of long cycle, many ELISA reaction steps, complicated process, etc., and achieve the effect of reducing harm, easy popularization and application, and reducing use.

Active Publication Date: 2021-02-26
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment of the double-antibody sandwich ELISA method requires the preparation and pairing of two antibodies, the process is complex, the cycle is long, and the ELISA reaction steps are numerous

Method used

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  • Cry1ab toxin mimetic antigen based on anti-idiotypic nanobody and its application
  • Cry1ab toxin mimetic antigen based on anti-idiotypic nanobody and its application
  • Cry1ab toxin mimetic antigen based on anti-idiotypic nanobody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of Natural Nanobody Library

[0037] (1) Extract the peripheral blood of unimmunized camels, separate lymphocytes and extract total RNA by TRIZOL;

[0038](2) Use cDNA synthesis via SuperScript III First-Strand Synthesis SuperMix Kit to synthesize cDNA by reverse transcription, and use nested PCR to amplify the nanobody gene (for PCR primers, see the literature "Ebrahimizadeh, W.; Gargari, S.M.; Rajabibazl, M.; Ardekani, S.; Zare, H.; Bakherad, H. Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes. Appl Microbiol Biotechnol. 2013, 97, 4457–4466").

[0039] The first round of PCR reaction system (50 μL) is: 1 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, 0.5 μL DNA polymerase, make up the balance with deionized water;

[0040] The first round of PCR reaction conditions are: 94°C 4min, 98°C 10s, 55°C 15s, 72°C 45s, 30 cycles, 72°C 7min;

[0041] The results of the f...

Embodiment 2

[0051] Example 2 Panning and Identification of Cry1Ab Mimic Antigen

[0052] (1) Panning of Cry1Ab mock antigen

[0053] Dilute the anti-Cry1Ab monoclonal antibody to 100 μg / mL with 10 mM PBS (pH 7.4), coat the Costar microtiter plate, and incubate overnight at 4°C; wash with PBST (pH 7.4 PBS, add 0.1% Tween-20 by volume) 3 times, add 300 μL of 3% BSA-PBS (3% OVA-PBS), block at 37°C for 2 h; wash 6 times with PBST, add 100 μL of camel-derived natural nanobody library prepared in Example 1 (titer about 2.0 ×10 11 pfu), incubated at 37°C for 1 h; washed 10 times with PBST, added 100 μL Glycine-HCl (0.2M, pH 2.2) to elute for 8 min, immediately neutralized with 15 μL Tris-HCl (1M, pH 9.0), and took 10 μL to wash The titer was measured by removing the phage, and the rest was used to infect 20mL of E.coli TG1 strain grown to the pre-logarithmic stage for amplification, and after purification, it was used for the next round of screening; in the subsequent three rounds of panning, ...

Embodiment 3

[0057] Example 3 Mass preparation of Cry1Ab mimic antigen

[0058] (1) Preparation by phage amplification

[0059] The positive phage clone cells obtained in Example 2 were inoculated in a 50mL 2×YT-AG medium Erlenmeyer flask, and cultured with shaking at 220rpm at 37°C until OD 600 =0.5; add helper phage M13K07, let stand at 37°C for 15 minutes, continue to culture at 37°C at 220 rpm for 45 minutes; centrifuge the culture at 10,000 rpm for 10 minutes, collect the cells, resuspend the cells in 50 mL of 2×YT-AK medium, and resuspend the cells at 30°C at 220 rpm Shake culture overnight; centrifuge the culture at 10,000 rpm at 4°C for 10 min, collect the supernatant, add 1 / 6 volume of PEG / NaCl, mix well and let stand at 4°C for 4 h; centrifuge at 10,000 rpm at 4°C for 10 min, discard the supernatant, and resuspend the precipitate Suspend in 1mL PBS, add an equal volume of glycerol and store at -80°C for later use.

[0060] (2) Preparation in the form of protein expression

[0...

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Abstract

The invention belongs to the field of genetic engineering antibody and food biotechnology, and relates to a Cry1Ab toxin mimic antigen based on an anti-idiotype nanobody and its application. The amino acid sequence of the Cry1Ab toxin mimic antigen is shown in SEQ ID NO.1, and the nucleoside encoding the amino acid The acid sequence is as described in SEQ ID NO.9, and the Cry1Ab mimic antigen provided by the present invention can replace the expensive and toxic Cry1Ab standard product, and is used as a competitive antigen in the competitive immunological detection of Cry1Ab, which has a molecular similarity to Cry1Ab Immune response properties, good results.

Description

technical field [0001] The invention belongs to the field of genetic engineering antibody and food biotechnology, and specifically relates to a Cry1Ab toxin mimic antigen based on an anti-idiotypic nanobody and an application thereof. Background technique [0002] Bt toxin is a parasporal crystal protein produced by Bacillus thuringiensis (Bt) when it forms spores, and can be divided into crystal protein (Cry protein) and extracellular soluble protein (cytolytic protein, Cyt protein) Two categories, which have insecticidal activity against various insects such as Lepidoptera and Coleoptera. The mechanism of its toxicity is that Bt protoxin is hydrolyzed into 60-70KD activated toxin under the action of insect midgut alkaline environment and protease, and then binds to the specific receptor on the midgut epithelial cell membrane, which leads to cell membrane perforation, and finally leads to Insects die. Bt toxin is highly specific to target insects, harmless to humans and a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/70C12N1/21C07K16/12G01N33/577G01N33/68C12R1/19
CPCC07K14/325C07K16/1278G01N33/577G01N33/68
Inventor 张存政邱雨楼刘贝贝王玉龙李盼刘贤金
Owner JIANGSU ACAD OF AGRI SCI
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