Nanobodies that can specifically bind to the c-myc tag
A nanobody and labeling technology, which is applied in the field of genetically engineered antibodies, single domain heavy chain antibodies or polypeptides, can solve the problems of limited sources of polyclonal antibodies, cumbersome and complicated monoclonal antibody development and production processes, etc.
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Embodiment 1
[0023] Construction of immune library of anti-c-Myc tag single domain heavy chain antibody (ie, single domain heavy chain antibody against c-Myc tag)
[0024] Covalently couple the c-Myc tag with Bovine serum albumin (BSA) to obtain the c-Myc artificial antigen c-Myc-BSA. After emulsifying 300μg of c-Myc-BSA with Freund’s complete adjuvant, The alpaca (Lama pacos) was immunized with multiple subcutaneous injections. The booster immunization was emulsified with 150μg c-Myc-BSA and Freund’s incomplete adjuvant, and the blood was collected 7 days after each immunization. The serum titer was determined by indirect ELISA, and the sample with the highest serum titer was selected to separate the lymph. Cell, extract RNA.
[0025] The RNA was extracted according to the instructions of TAKARA's RNAiso reagent. Using RNA as template and oligo dT as primer, the first strand of cDNA was synthesized according to the reverse transcriptase instructions of TAKARA Company.
[0026] PrimeSTAR high-...
Embodiment 2
[0034] Panning and identification of anti-c-Myc tag single domain heavy chain antibody
[0035] A solid phase affinity panning method was used to pan the single domain heavy chain antibody against the c-Myc tag from the anti-c-Myc tag single domain heavy chain antibody immune library obtained in Example 1. Add 120 μL of Myc-GST fusion protein (a protein fused with Myc tag and glutathione) diluted with PBS to each microtiter plate, and coat overnight at 4°C. The coating concentration for each round of panning is 100. 75,50μg / mL; aspirate the coating solution, wash the plate with PBS 5 times, add 300μL 3% BSA-PBS to each well, block for 2h at 37°C; wash the plate with PBS 5 times, add 100μL phage antibody library (about 1×10 11 CFU), 37℃, incubate for 2.0h; aspirate unbound phage, wash the plate 3-5 times with PBST (containing 0.5% Tween-20) (increase 5 times in each round), and then wash the plate 15-25 times with PBS; Use 100μL of eluate (glycine-hydrochloric acid, pH 2.2) to elu...
Embodiment 3
[0045] Large-scale preparation of anti-c-Myc tag single domain heavy chain antibody
[0046] Obtaining the DNA fragment encoding the anti-c-Myc tag single domain heavy chain antibody: 1. Using restriction endonucleases SfiI / NotI, double digestion of the phagemid pHEN-anti-c-Myc single domain heavy chain antibody gene, agar Glycogel electrophoresis to recover the anti-c-Myc tag single domain heavy chain antibody gene; 2. Directly send the anti-c-Myc tag single domain heavy chain antibody coding sequence to the biotechnology service company for chemical synthesis; 3. Design specific primers and pass PCR technology was used to amplify from a cDNA library derived from alpaca (Lama pacos).
[0047] The obtained anti-c-Myc tag tag single domain heavy chain antibody gene fragment was cloned into the expression vector pET25-flag (the c-Myc tag of the vector itself has been replaced with the Flag tag: DYKDDDDK), and it was constructed after PCR and restriction enzyme digestion. Complete th...
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