A kind of epitope peptide simulating dienestrol, its preparation method and application
An epitope, diethylstilbestrol technology, applied in the direction of peptides containing affinity tags, peptides containing MBP tags, peptides, etc., can solve the adaptive matching of antigenic epitope peptide immunological properties, difficult to obtain detection effect, lack etc.
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Embodiment 1
[0035] Example 1, Panning and Identification of DIS Antigen Mimic Epitopes
[0036] (1) Affinity panning of DIS antigen mimotope: the specific method is: use 0.1M NaHCO 3 (pH 8.6) dilute the anti-DIS monoclonal antibody, and add it to a 96-well enzyme-labeled single well at a final concentration of 80 μg / ml, and coat overnight at 4°C. The next day, after rapid washing 6 times with TBST [50mM Tris-HCl (pH7.5), 150mM NaCl, 0.1% Tween-20 (v / v)], 350μl of 1% OVA blocking solution was added, and blocked at 4°C for 2h. Discard the blocking solution, wash 6 times with TBST, add 120 μl phage peptide library (phage display heptapeptide library, purchased from NEB Company, dilute the phage with TBST, the addition amount is 2.0×10 11 pfu), react at 25°C for 50min. The phage in the wells were discarded, quickly washed 10 times with TBST, patted dry, and eluted with 0.2M Glycine-HCl (pH 2.2) for 8 min, and then neutralized with 15 μl 1MTris-HCl (pH 9.1). Take 5 μl of the eluted phage to...
Embodiment 2
[0039] Example 2, Sequencing of DIS Antigen Mimotope Encoding Gene and Determination of its Amino Acid Sequence
[0040] The phage identified as the mimotope of the DIS antigen by indirect competitive ELISA was amplified, and the DNA of the phage was extracted. The operation process is as follows: amplify the target phage, centrifuge the amplified product, transfer 500 μl phage supernatant to a new centrifuge tube; add 200 μl PEG / NaCl to precipitate the phage, let stand for 20 minutes, and centrifuge at 14000 rpm for 10 minutes. Discard the supernatant, resuspend the pellet in 100 μl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4M NaI), add 250 μl absolute ethanol to precipitate, let stand for 15 min, and centrifuge at 14000 rpm for 10 min. After the supernatant was discarded, the precipitate (DNA sequencing template) was washed with 70% ethanol, centrifuged at 14000 rpm for 10 min, the supernatant was discarded, and briefly dried in vacuum. Resuspend the pellet in 30μl...
Embodiment 3
[0041] Example 3, Application of DIS Antigen Mimotope as Competing Antigen in Enzyme-Linked Immunoassay Method
[0042] (1) Sample extraction
[0043] Remove the fat and connective tissue of the pork, break it with a mixer, weigh 2g into a 50ml centrifuge tube, add 6ml of 10% methanol-PBS solution, shake on the shaker for 3min; add 6ml of 0.05M carbonate Buffer solution (pH9.6), then add 10ml of ethyl acetate, shake at 150rpm for 30min, and centrifuge at 4000rpm for 10min. Pipette the upper organic layer into another test tube, add 1.25ml 0.1M NaOH solution and 0.625ml 1.0M MgCl to it 2 solution in a water bath at 60°C for 30 minutes. Take out the test tube and add 1g of anhydrous Na after cooling for a while 2 SO 4 , Shake on a shaker for 5 minutes and then centrifuge for 5 minutes. Transfer the upper organic layer to a new test tube, and dry it in a nitrogen blower at about 55°C. Add 100 μl of methanol solution to dissolve, then add 900 μl of PBS to resuspend, and filt...
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