A Cry2ad-binding Cyclic Heptapeptide and Its Encoding Gene and Application
A technology encoding gene and cyclic heptapeptide, applied in the field of cyclic heptapeptide, can solve the problem of short shelf life of antibodies, achieve the effect of good detection effect, wide application prospect and simplified preparation process
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Embodiment 1
[0042] Example 1. Panning and Identification of Cyclic Heptapeptides Binding to Cry2Ad
[0043] 1. Specific method for affinity panning of cyclic heptapeptide combined with Cry2Ad:
[0044] a. The first round of panning: Coat a 6-well ELISA plate with 100 μg / mL BSA and 100 μg / mL Cry2Ad, and incubate overnight at 4°C. The next day, after washing 6 times with 0.1% (v / v) Tween-20 (Tween-20) TBST, pre-screen with BSA as the negative selection target, then bind to the activated Cry2Ad toxin, wash 10 times with 0.1% TBST after binding , and eluted by competitive elution. The eluate was infected with 20mL logarithmic phase E.coil ER2738 for 4.5h amplification, and the phage was precipitated overnight with PEG / NaCl, centrifuged and resuspended the next day to obtain a secondary library.
[0045]b. Further screening: Repeat step a for three more rounds of panning. In the second to fourth rounds of panning, the coating concentrations of Cry2Ad were 75, 50, and 25 μg / mL, respectively, ...
Embodiment 2
[0052] Example 2. Specific detection of cyclic heptadeptide binding to Cry2Ad
[0053] The optimal coating concentration of Cry2Ad and the optimal dilution factor (10) of positive phage clones were determined by checkerboard titration. 12 、10 11 、10 10 、10 9 、10 8 pfu). Coat the microwells with the optimal Cry2Ad concentration, overnight at 4°C, and after blocking with MPBS, add the mixture of toxin and polypeptide incubated overnight (take 50 μL of phage-positive clones with the optimal dilution factor and mix with 50 μL of Cry2Ad toxin, the toxin concentration is 320 μg / mL starting at 4 times and down to 8 gradients), at the same time BSA was used as a negative control, HRP-labeled anti-M13 was added, and A was determined after TMB color development 450 value. Repeat the experiment 3 times in parallel to calculate the inhibition rate. Inhibition rate calculation method: inhibition rate = (A before inhibition 450 - After inhibition A 450 ) / Suppression Pre-A 450 ×10...
Embodiment 3
[0056] Example 3. Establishment of a sandwich ELISA method using a Cry2Ad-binding cyclic heptapeptide as a detection element
[0057] (1) Mass production of specific cyclic heptapeptides by means of phage amplification
[0058] The phage displaying the specific cyclic heptapeptide was added to 20 mL of ER2738 cultured to the logarithmic growth phase, and cultured at 37° C. for 4.5 h. Centrifuge at 12,000 g for 10 min, add 1 / 6 volume of PEG / NaCl to 16 mL of the supernatant, let stand at 4°C for 1 h, centrifuge at 10,000 rpm for 15 min, and resuspend the pellet in 1 mL of TBS to obtain the amplification solution.
[0059] (2) Establishment of standard curve
[0060] The optimal coating concentration of BBMV and the optimal dilution factor of positive phage clones were determined by checkerboard method. BBMV coating concentration is 10μg / mL phage dilution is 10 11 pfu. After the BBMV was coated overnight, it was washed 3 times with PBST, and blocked with 1% BSA for 1 hour at ...
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