Anti-TIGIT nanobody and application thereof
A nanobody, expression vector technology, applied in the field of biomedicine or biopharmaceuticals
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Embodiment 1
[0187] Example 1: Human TIGIT extracellular protein expression and camel immune library construction
[0188] Use mammalian cell HEK293F to transiently express human TIGIT extracellular domain protein: mix the pFUSE-IgG recombinant plasmid that has cloned human TIGIT extracellular domain gene with transfection reagent PEI, and then transfect into HEK293F cells; 37°C, 6% CO 2 Cultivate in a shaker incubator for 5 days; then collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; wash the beads with phosphate buffer solution pH 7.0, and elute the protein with 0.1M pH 3.0 glycine solution; The eluted protein was ultrafiltered into PBS solution, and after the yield was measured, samples were taken for SDS-PAGE detection. Test results such as figure 1 As shown, the expressed and purified hTIGIT(ECD)-Fc protein has a purity greater than 90%, which can be used for camel immunization and antibody screening.
[0189] The purified hTIGIT(ECD)-Fc p...
Embodiment 2
[0191] Example 2: Screening of blocking TIGIT Nanobodies by flow cytometry
[0192] Cloning the Nanobody genes with different sequences from the above strains into the pFUSE-IgG vector, and then mixing the recombinant plasmid with the transfection reagent PEI 1:3, transfected into HEK293F cells; 37°C, 6% CO 2 Cultivate in a shaker incubator for 5 days; then collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; wash the beads with phosphate buffer, pH 7.0, and elute the protein with 0.1M, pH 3.0 glycine solution; The eluted protein was then ultrafiltered into PBS solution for candidate functional activity research.
[0193] Aliquot the cultured CHOZEN / TIGIT stably transfected cells into 96-well plates, 3E5 cells per well, centrifuge at 3000rpm for 3min to remove the supernatant, add diluted antibodies (Nb5, Nb10, Nb14, Nb16, Nb49, Nb66 , Nb72, Nb86, Nb96, Nb100) and CD155-Biotin protein were incubated for 20 min, and the antibody concent...
Embodiment 3
[0194] Example 3: Detection of binding activity of TIGIT nanobody to cell surface antigen
[0195] Antibody binding function verification was performed using CHOZN / TIGIT stably transfected cells with high expression of TIGIT: the cultured CHOZN / TIGIT cells were digested with trypsin and neutralized with complete medium, and the cells were washed once with PBS, and then the cells were collected; In the 96-well plate, add the diluted antibody (the dilution concentration of each group of antibodies is 40ug / mL, 20ug / mL, 10ug / mL, 5ug / mL, 2.5ug / mL, 1.25ug / mL, 0.625ug / mL, 0.313 ug / mL, 0.156ug / mL, 0.078ug / mL, 0.039ug / mL, 0.019ug / mL) were incubated at 4°C for 20min; after centrifugation, the cells were washed once with PBS, and then goatanti human IgG-FITC was added (according to 1:200 Dilute), incubate at 4°C for 20min; wash the cells once with PBS, centrifuge at 3000rpm, 4°C for 4min, discard the supernatant, add 200uL PBS / well, resuspend the cells, transfer to a flow tube, and detec...
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