CD47 nanobody and application thereof
A single-domain antibody, expression vector technology, applied in the field of biomedicine or biopharmaceuticals, can solve the problems of harsh storage conditions and low stability
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Embodiment 1
[0132] Example 1: Expression and purification of human CD47 protein
[0133] (1) The nucleotide sequence of human CD47 was synthesized on the pCDNA3.1(-) vector, and then its extracellular segment sequence was subcloned into the pFUSE-IgG1 vector; (2) The Omega plasmid extraction kit was used to extract and construct pFUSE-IgG1-hCD47 (ECD) plasmid; (3) Culture HEK293F cells to an OD of 2.0×10 6 cells / mL; (4) Mix the plasmid and transfection reagent PEI 1:3 evenly, let it stand for 20min, and then add it to HEK293F cells at 37°C, 6% CO 2 Cultivate in a shaker incubator for 5-6 days; (5) collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; (6) wash the beads with phosphate buffer solution pH 7.0, and then use 0.1M pH 3.0 Glycine Elution protein; (7) ultrafilter the eluted protein into PBS, take a sample after measuring yield and carry out SDS-PAGE detection (detection result such as figure 1 shown), and the rest of the protein was store...
Embodiment 2
[0134] Example 2: Construction and screening of CD47 single domain antibody library
[0135] Library construction: Briefly, (1) mix 1 mg hCD47(ECD)-Fc antigen with Freund's adjuvant in equal volume, immunize a Xinjiang Bactrian camel once a week, immunize 7 times in total, and stimulate B cells to express antigen-specific (2) After 7 times of immunization, extract 100mL camel peripheral blood lymphocytes and extract total RNA; (3) Synthesize cDNA and amplify VHH by nested PCR; (4) Use restriction endonuclease 20 μg pMECS phage display vector (supplied by Biovector) and 10 μg VHH were digested with Pst I and Not I enzymes, and the two fragments were ligated; (5) The ligated product was transformed into electroporation-competent cells TG1 to construct a CD47 single-domain antibody library and measure the library capacity, Storage capacity is 2.5×10 9 CFU (results such as figure 2 shown). At the same time, 24 clones were randomly selected for colony PCR detection, and the res...
Embodiment 3
[0137] Example 3: Expression and purification of CD47 single domain antibody in eukaryotic cells HEK293 and detection of blocking function of single domain antibody by flow cytometry
[0138]Eukaryotic cell HEK293F expresses CD47Nb-Fc fusion protein: (1) Cloning the CD47Nb sequence with correct sequencing results into pFUSE-IgG4 vector (purchased from Invivogen), and extracting pFUSE-IgG4-Nb plasmid with Omega plasmid extraction kit; (2) ) to culture HEK293F cells to OD of 2.0×10 6 cells / mL; (3) Mix the plasmid and the transfection reagent PEI at a ratio of 1:3, let it stand for 20 minutes, and then add it to HEK293F cells at 37°C, 6% CO 2 Cultivate in a shaker incubator for 5-6 days; (4) collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; (5) wash the beads with phosphate buffer solution pH 7.0, and then use 0.1M pH 3.0 Glycine Elution protein; (6) the protein ultrafiltration of elution is entered in PBS, after measuring output, take...
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