Semi-synthetic nano antibody library construction method and nano antibody library
A nanobody and construction method technology, applied in chemical instruments and methods, libraries, microbial libraries, etc., to improve efficiency, save costs and time
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Embodiment 1
[0071] The examples of this application provide a method for constructing a semi-synthetic nanobody library, such as figure 1 As shown, the method includes the following steps:
[0072] S1. respectively obtain the cDNAs of CDR1, CDR2 and CDR3 as templates;
[0073] S2. Obtain the nucleotide sequences of FR1, FR2, FR3 and FR4 respectively;
[0074] S3. Using the cDNAs of CDR1, CDR2 and CDR3 as templates, carry out the first amplification to obtain the base sequences of CDR1, CDR2 and CDR3 respectively;
[0075] S4. connect the nucleotide sequences of FR1, FR2, FR3 and FR4 with the nucleotide sequences of the CDR1, the CDR2 and the CDR3, and perform a second amplification to obtain the target gene fragment;
[0076] S5. the target gene fragment is connected with the first carrier to obtain the second carrier containing the target gene fragment;
[0077] S6. Cultivate the second vector so that the target gene fragment is expressed as a nanobody, and then screen to obtain a tar...
Embodiment 2
[0090] Example 2 Rescue of phage surface-displayed nanobody libraries
[0091] The nanobody library constructed in Example 1 is stored in the host bacteria in the form of phagemids. Before the panning process starts, the library should be rescued to make it a phage-displayed nanobody library. The specific method is as follows:
[0092] 0.4;37 shake culture about 1.5h to OD 600nm = 0.5-0.6; add helper phage (M13K07) according to bacteria:phage = 1:5, shake and culture at 37 for about 1 h; centrifuge at 4000 rpm for 15 min, remove the medium; add 200 mL of 2YT-AK (100 μg / mL Amp, 50 μg / mL) mL Kan) medium to resuspend the bacteria, culture for 2h at 37; centrifuge at 10,000 rpm for 20 min to remove the precipitate; add 40 mL of PEG / NaCl to the supernatant to precipitate the phage, and ice-bath overnight; centrifuge at 10,000 rpm for 20 min, remove the supernatant; resuspend the phage with 0.6 mL of 2YT medium, 4 spares. If large-scale production of phage is required, after ch...
Embodiment 3
[0094] Example 3 Antibody Screening
[0095] 1) Using His Bind Resin to bind antigen proteins, panning antibodies from the phage-displayed nanobody library obtained in Example 2. The specific process is as follows: Activating His Bind Resin: Take 200 μL of His Bind Resin in a 1.5 mL centrifuge tube, and centrifuge at 1000 g for 1 min , remove the preservation solution; add 200 μL of ddH 2 O wash the resin once, centrifuge at 1000g for 1 min, remove the supernatant, and repeat this step once; add 200 μL of ionization buffer, resuspend the resin, let stand for 10 min, and remove the supernatant by centrifugation; add 200 μL of binding buffer, resuspend the resin, and let stand 10min; 40μL of resin was added to a 1.5mL centrifuge tube, and the supernatant was removed by centrifugation.
[0096] 2) Panning for Nanobodies that recognize the target protein
[0097] Take 35 μL (about 10 μg) of purified PG1 protein, add it to 165 μL PBS, add it to the EP tube containing the activate...
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