Polypeptide molecules capable of specifically binding to deoxynivalenol and applications thereof
A deoxynivalenol and specific technology, which is applied to peptides, analytical materials, instruments, etc., can solve the problems of no amino group, carboxyl group on the surface, difficult polypeptide molecule binding, and small size of small molecular substances, etc., to achieve Good structural stability and simple preparation
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Embodiment 1
[0015] Example 1. Affinity panning and identification of polypeptide molecules specifically binding to DON
[0016] 1) The specific method for affinity panning and DON-specific binding polypeptide molecules is as follows: take 0.5 mg of DON-OVA whole antigen (coupling ratio is 15:1), and coat it on the wells of the microtiter plate (100 μL / well), 37 Incubate at °C for 2 hours. After washing 10 times with PBST (10 mM PBS pH 7.4 containing 0.1% Tween-20 (v / v)), 350 µl of blocking solution (5% gelatin solution) was added and incubated at 37°C for 2 hours. Wash 5 times with PBST, add 100 μl phage peptide library (phage display cyclic heptapeptide library, purchased from NEB Company, dilute phage stock solution 10 times with PBS, about 2.0×10 11 pfu), shaken at 37°C for 2 hours. Unbound phages were discarded, washed 15 times with PBST, and bound phages were eluted with 100 ng of DON antibody dissolved in PBS. Take 10 μl of the eluted phage to measure the titer, and the rest is u...
Embodiment 2
[0020]Example 2. Sequencing of the coding gene for a polypeptide molecule specifically binding to DON and determination of its amino acid sequence
[0021] 1. Amplify the phages that display specific binding to DON polypeptide molecules identified by indirect competition ELISA, and extract the DNA sequencing templates of the phages. The brief process is as follows: carry out phage amplification, after the first step of centrifugation, transfer 800 μl of phage-containing supernatant into a new centrifuge tube. Add 200 μl PEG / NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100μl iodide buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), add 250μl absolute ethanol to precipitate DNA, and wash the pellet with 70% ethanol after centrifugation (DNA sequencing template ). The pellet was finally resuspended in 20 μl sterilized water, and 2 μl was taken for agarose gel electrophoresis analysis; 5 μl of phage template was taken for DNA sequencing, and its -...
Embodiment 3
[0022] Example 3. Application of specific binding DON polypeptide molecules in ELISA
[0023] (1) Sample extraction
[0024] Weigh 5g of the sample to be tested, add 25ml of PBS solution, and shake at 200rpm for 5 minutes; filter the extract with Whatman No. 1 filter paper, take 1ml of the filtrate and add 1ml of PBS (phosphate buffer saline, pH=7.2) and mix well. It is the sample extract, ready for use.
[0025] (2) Coating and sealing
[0026] DON-OVA (2 μg / ml) diluted with 10 mM PBS (pH 7.4) was used to coat the microtiter plate and incubated overnight at 4°C. The next day, after washing 3 times with PBST (10mM PBS, 0.05% Tween-20 (v / v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, wash the plate 6 times with PBST until use.
[0027] (3) Establishment of standard curve
[0028] Take out the strips treated in step (2), and put 50 μl of phage (1.0 × 10 12 pfu) and a series of 50 μL DON standard (0-10,000 ng / ml) at different concentrati...
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