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
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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...
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