CD147-resistant nanometer antibody and preparation method and application thereof
A nanobody and antibody technology, applied in the fields of molecular biology and immunology, to achieve low heterogeneity, small steric hindrance, and strong targeting
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Embodiment 1
[0046] Example 1. Panning of anti-CD147 protein nanobody, identification and sequencing of positive clones
[0047] Using the method of solid-phase panning, dilute CD147 protein to 30-100ug / ul with 1xPBS, coat it on the ELISA strip, add 100ul to each well, and coat overnight at 4°C. Wash three times with PBS, add 300ul 4% skimmed milk to each well, and block for 2 hours at 37°C. After washing three times with PBS, add the phage display nanobody library (about 1x10 12 CFU), 37°C for 1 hour. Aspirate the unbound phage, add PBS to wash 5-10 times (increase the number of washes with each round), and then wash three times with PBST, then add 100ul of glycine-hydrochloric acid solution with pH=2.2, 37°C for 7 minutes. Gently blow the wells of the plate to wash off the adsorbed phages, then add 15ul of Tris-HCl solution (PH=8.8), take 10ul to measure the titer, and the rest will be amplified and used for the next round of panning.
[0048] After three rounds of panning, clones wer...
Embodiment 2
[0056] Example 2, Expression and Purification of Nanobodies
[0057] The above nucleotide sequence was inserted into the PET15b plasmid to construct an expression vector. Take 2ul of the constructed PET15b plasmid and add it to 50ul of BL21 competent cells, place it on ice for 30 minutes, then place it in a water bath at 42°C for heat shock for 90 seconds, place it on ice for 10 minutes, and add 800 μL of non-resistant LB to the suspension Liquid culture medium, after mixing, incubate at 37°C with shaking at 150rpm for 1h, centrifuge at 3500rpm at room temperature for 4min, carefully aspirate and discard 700μL supernatant, leave about 200μL medium, repeatedly blow and beat the suspended bacteria, and spread on LB-Amp solid plate ; Place the plate in a constant temperature incubator at 37°C, culture it upright for 10 minutes, and then culture it upside down overnight. The next day, single clones were picked from the overnight culture plate, placed in 10 mL LB-Amp liquid medium...
Embodiment 3
[0059] Example 3, Western blot verification of Nanobodies
[0060] 1. Labeling of Nanobody HRP
[0061] Take 40ug of antibody solution (calculate the volume according to the concentration), add 12ul (100μL=1mg) REAGENT Ⅰ A type activated horseradish oxidase. Use REAGENT Ⅱ to adjust the pH value to about 9.5 (about 10ul, the amount is different due to the difference in the buffer used for the antibody, but ensure that the pH is above 9.0, you can add more REAGENT Ⅱ, and accurately record the usage of REAGENT Ⅱ for future use. One step), 37°C, 30min. A small amount of sodium borohydride was added. (Suitable for long-term storage of enzyme-labeled substances, no need to add for immediate use. Take a 200ul pipette tip, insert sodium borohydride powder, white powder can be seen at the tip of the tube, then transfer to enzyme-labeled substances, pipette to dissolve and mix well ). Add REAGENT Ⅲ (about 3 times the volume of REAGENT Ⅱ). 10ul of REAGENT Ⅱ was used in the previous ...
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