Coding gene of drosophilid MYC nano antibody, preparation method and application thereof
A technology of nano-antibody and coding gene, applied in the field of genetic engineering, can solve the problems of high production cost, affecting the application of MYC antibody, poor stability, etc., and achieve the effects of small molecular weight, easy penetration into tissue cells, high specificity and affinity
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Embodiment 1
[0033] Example 1: Construction of alpaca nanobody library after Drosophila MYC immunization
[0034] (1) Drosophila embryo protein lysate was mixed with an equal volume of adjuvant (purchased from GERBU), and an alpaca was immunized once every two weeks for a total of 3 times.
[0035] (2) After the 3 times of immunization, 50 ml of peripheral blood of the alpaca was extracted, the blood lymphocytes were separated by density gradient centrifugation (dextran-diatrizoate), and the total RNA of the lymphocytes was extracted by the Trizol method.
[0036] (3) According to TAKARA's PrimerScript TM Instructions for the 1st Strand cDNA Synthesis Kit, which reverse-transcribes the extracted RNA into cDNA.
[0037] (4) Utilize nested PCR (the enzyme used in PCR is TAKARA company Max DNA Polymerase) to amplify the VHH region of alpaca heavy chain antibody. The first round reaction conditions are: 98°C, 3min; 98°C, 10s, 55°C, 10s, 72°C, 1min, 30 cycles; 72°C, 10min. The PCR product...
Embodiment 2
[0041] Example 2: Enrichment process of Drosophila MYC protein-specific nanobodies
[0042] (1) According to the method of prokaryotic purification, the MYC protein fused with the His tag was purified, and the result figure 1 shown.
[0043] (2) Take 1 μg of purified MYC protein and incubate with 30 μl of His-coupled magnetic beads at room temperature for 30 min to bind MYC protein to the magnetic beads, and then wash with PBST for 3 times to wash away unbound MYC protein.
[0044] (3) Add 500 μl phage library (containing 5×10 12 A phage displaying the immune alpaca nanobody), and incubated at room temperature for 2h. Wash 25 times with PBST to wash away unbound or weakly binding phages.
[0045] (4) 500 μl trypsin (0.25 mg / ml) was used to dissociate the phages specifically bound to MYC, and 10 μl protease inhibitor cocktail (purchased from Roche Company) was added to the dissociated phage solution for neutralization.
[0046] (5) Take 300 μl of the dissociated phage solutio...
Embodiment 3
[0048] Example 3: Enzyme-linked immunosorbent assay (ELISA) screening of MYC-specific nanobody positive monoclonal
[0049] (1) Randomly select 10 bacterial monoclonal clones from the overnight cultured Nanobody bacterial library obtained after the third round of screening above, inoculate them into LB medium respectively, and cultivate them until the logarithmic phase of bacterial growth, adding a final concentration of 0.2mM IPTG was incubated overnight at 30°C to induce Nanobody expression.
[0050] (2) The bacteria were collected the next day, and CelLytic was used to TM B Cell Lysis Reagent (purchased from Sigma Company) was used to lyse the bacteria to obtain the nanobody crude extract. Take 100 μl antibody crude extract and add to the ELISA plate coated with MYC protein and blocked with 3% BSA, and incubate at room temperature for 1 h.
[0051] (3) Wash 3 times with PBST, 2 min each time, to wash away unbound protein.
[0052] (4) Anti-pIII antibody (1:1000, purchase...
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