Anti-4-1BB nano antibody, coding gene and application
A nanobody, encoding gene technology, applied in the direction of antibodies, applications, genetic engineering, etc., to achieve the effect of wide application prospects
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Embodiment 14-1
[0066] Example 1 Expression and purification of 4-1BB protein
[0067] 1.1 Vector construction
[0068] The eukaryotic expression vector pFuse was used to construct the 4-1BB-Fc fusion protein, and 4-1BB and human IgG1 Fc were connected in series by PCR. The complete molecule included 4-1BB secretion signal peptide, 4-1BB extracellular domain, thrombin cleavage site, IEGRMD short peptide, human IgG1 hinge region, and CH2 and CH3 domains. The 4-1BB-Fc gene was double digested with NcoI and NheI and then cloned into the pFuse vector using T4 DNA ligase, or a homologous recombination kit was used to construct a vector.
[0069] 1.2 Eukaryotic expression
[0070] Inoculate 1.5×10 6 / mL 293F cells in 200mL culture medium in a 500mL shake flask. 37°C, 165rpm, 5% CO2 concentration in a shaking incubator for 24h, count the cells after 24h, and adjust the cell density to 3×10 6 / mL, add 200μg of the plasmid constructed in step 1.1 above to 10mL opti-MEM and mix gently, then let st...
Embodiment 2
[0077] Example 2 Construction and Screening of Anti-4-1BB Nanobody Phage Display Library
[0078] 2.1 Immunity of alpacas
[0079] Choose a healthy adult alpaca and mark the ear number. Mix 4-1BB protein with Freund's adjuvant at a ratio of 1:1 and store at 4°C. The left and right sides were injected near the lymph nodes in the alpaca neck, 2 points on each side, and 0.4 mL of mixed adjuvant antigen was injected each time. Observe for half an hour after immunization to confirm that the alpaca is in good condition and has no adverse reactions. Immunization was performed every 2 weeks, and a total of 7 immunizations were performed. At intervals of 5-7 days after the 6th and 7th immunization, the peripheral blood of the alpaca was collected from the neck vein for the construction of the phage display library.
[0080] 2.2 Isolation of lymphocytes derived from alpaca
[0081] Add 3 mL of cell separation medium to a 15 mL centrifuge tube, and slowly add 3 mL of blood sample. ...
Embodiment 3
[0151] Example 3 Sequencing
[0152] 3.1 Positive clone sequencing
[0153] Positive monoclonals were selected based on ELISA detection data and secondary verification data.
[0154] Take 5 μL of the positive clone liquid from the monoclonal ELISA detection plate and inoculate it into 2 mL of 2×YT medium (containing 100 μg / mL Amp and 10 μg / mL Tet), culture at 37°C, 250 rpm until OD 600 To 0.8-1.0 (about 6-8h), take 1mL of the bacterial liquid for sequencing, and store the rest of the bacterial liquid at 4°C.
[0155] 3.2 Sequence Analysis
[0156] The sequenced sequences were analyzed using GENtle software for sequence alignment, and the antibody sequences were translated into amino acids using GENtle software. The result is as Figures 7a-7e ,in Figure 7a shows the overall sequence similarity in the listed examples, Figure 7b~7e Sequence similarity between groups is shown.
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