Preparation and application of a nanobody specifically recognizing fenitrothion
The technology of nanobody and phosphalt, which is applied in the biological field, can solve the problem of not being provided phosphato, and achieve the effects of high organic tolerance and weak acid resistance, simple operation and universal applicability.
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[0059] 1. Preparation of immune antigen and detection antigen
[0060] The design and synthesis of the fenitrothion hapten was completed by the preliminary work of the laboratory, and two types of hapten structures were used in this experiment. Hapten structure A is used for the preparation of artificial immunization antigen, and hapten structure B is used for the preparation of artificial detection antigen. The structure of the above-mentioned hapten and artificial antigen is as follows: figure 1 shown.
[0061] Hapten A was coupled with lactoferrin (Lactoferrin, LF) by active ester method to form immune antigen. The specific operation method is as follows: Weigh 7.3mg EDC and 4.3mg NHS, add 0.1mL DMF to dissolve the solids. Add 9.7 mg of fenitrothion hapten A to the above mixture solution, stir or shake at room temperature in the dark for 4 hours to activate the hapten. Weigh 50mg LF, add 5mL carbonate buffer (pH 9.4) to prepare 10mg / mL protein solution. Add the activat...
Embodiment 2
[0097] Example 2 Affinity panning and identification of nanobodies
[0098] 1. Experimental method
[0099] 1. Affinity panning of nanobodies
[0100] (1) Detection of antigen immobilization
[0101] The detection antigen B-BSA was diluted to 1 μg / mL with PBS, added to the microwells of the microplate, 100 μL per well, and left at 4°C overnight. The next day, after washing the plate twice with PBST (0.01M PBS, 0.05% Tween-20), 150 μL of 1% BSA-PBS (w / v) solution was added to each well and allowed to stand at 37° C. for 1 h. Pour off liquid in wells and pat dry on absorbent paper for later use.
[0102] (2) Positive phage screening
[0103] Add BSA to the phage library in Example 1 so that the final concentration of BSA is 1% (w / v), add the mixture to 3 microwells with immobilized antigen, add 100 μL to each well, and incubate at 37° C. for 1 h. The free phage in the wells were discarded, the microwells were washed 10 times with PBST, and then washed 5 times with PBS. Add...
Embodiment 3
[0127] Example 3 Expression of Nanobodies as Single Domain Antibodies
[0128] The Ecoli.TG1 strain carrying the expression vector of the nanobody sm6 (amino acid sequence shown in SEQ ID NO.1) gene after gene sequencing was extracted with a kit, and transformed into competent E.coil BL21 (DE3) And spread on LB-Agar-Amp plate culture to obtain a single colony. Pick a single colony and inoculate it in 10mL LB-Amp, and culture overnight at 37°C and 250rpm. The overnight culture was inoculated into 100mL LB-Amp at a ratio of 1:100, cultured at 37°C, 250rpm to the logarithmic phase, added IPTG to the working concentration, and cultured overnight at 37°C, 250rpm. Centrifuge the bacterial solution and discard the supernatant, redisperse the pellet in 10mL PBS, freeze at -80°C for 30min, and thaw in a water bath at 37°C, repeat this process 3 times, sonicate for 30min, shake at 250rpm for 2h to fully extract the protein, and centrifuge to save the supernatant , the supernatant was ...
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