Anti-CCR8 monoclonal antibody and application thereof
A technology for monoclonal antibodies and applications, applied in the field of biomedicine or biopharmaceuticals, can solve the problems of lack of CCR8 monoclonal antibodies, small molecular weight, stability, immunogenicity and weak penetrability, etc.
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Embodiment 1
[0153] Example 1: Expression and purification of human CCR8 protein
[0154] The nucleotide sequence of human CCR8 was synthesized in a commercially available vector, and the prepared plasmid was evenly mixed with the transfection reagent PEI 1:3, then allowed to stand for 20 minutes, and then added to appropriate cells (HEK293F cells), at 37°C, 6 %CO 2 Cultivate in a shaker incubator for 5-6 days; collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; wash the beads with phosphate buffer solution pH7.0, and then use 0.1M pH3.0Glycine to elute the protein;
[0155] Ultrafilter the eluted protein into PBS, measure the yield and take a sample for SDS-PAGE detection, and store the rest of the protein in a -80°C refrigerator;
Embodiment 2
[0156] Example 2: Preparation of CCR8 monoclonal antibody
[0157] In this example, a fully human antibody yeast screening platform was used to screen CCR8-specific monoclonal antibodies through CCR8+CHO cell stable transfection.
Embodiment 3
[0158] Preparation of Example 3 Nanobodies
[0159] A certain amount of hCCR8 antigen was mixed with an equal volume of Freund's adjuvant, and an alpaca was immunized once a week for several times in total to stimulate B cells to express antigen-specific nanobodies. After the immunization, 100mL peripheral blood lymphocytes were extracted and total RNA was extracted; cDNA was synthesized and VHH was amplified by nested PCR; and a phage display library was constructed.
[0160] Through screening and identification, multiple strains of nanobodies were obtained.
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