Cry1Ab toxin simulated antigen based on anti-idiotype nanometer antibody and application thereof
A nano-antibody and anti-idiotype technology, which is applied in the field of genetic engineering antibodies and food biology, can solve the problems of ELISA reaction steps, complicated process, and long cycle, and achieve the effects of easy promotion and application, reduced harm, and reduced use
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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
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