Anti-PD-1 (programmed cell death-1) humanized antibody
A PD-1, human antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problem of only 10% single drug effective rate, achieve obvious affinity, inhibit ligand - effect of receptor binding
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Embodiment 1
[0041] Expression and purification of human PD-1 extracellular domain recombinant protein PD-1-His
[0042] Synthetic human PD-1-AP-His fusion gene (synthesized by Nanjing GenScript Biotechnology Co., Ltd.), wherein the PD-1 gene sequence is SEQ ID NO: 5, the AP-His gene sequence is SEQ ID NO: 6, two The connection of the segment sequences is the PD-1-AP-His fusion gene. For the construction of the PD-1-His recombinant protein expression vector, the forward and reverse primers are designed as follows:
[0043] Forward primer:
[0044] 5'-ATCCGCCGGCAAGCCGCCACCATGCAGATCCCACAGGCGCCCTGG-3' (SEQ ID NO: 7);
[0045] Reverse primer:
[0046] 5'-TCACTACGTATCAGTGGTGGTGGTGGTGGTGTTGGAACTGGCCGGCTGGCCTGG-3'(SEQ ID NO:8), used to amplify the gene encoding PD-1-His fusion protein, and introduce restriction enzyme sites Ngo MIV and Sna BI at the upstream and downstream of the gene respectively , PCR conditions are 95°C for 30s, 60°C for 30s, 72°C for 30s, 35 cycles, 72°C extension for 10mi...
Embodiment 2
[0049] Expression and purification of human PD-1 extracellular region recombinant protein PD-1-AP-His
[0050] Using the PD-1-AP-His fusion gene synthesized in Example 1 as a template, the forward and reverse primers were designed as: 5'-ATCCGCCGGCAAGCCGCCACCATGCAGATCCCACAGGCGCCCTGG-3'(SEQ ID NO:7) and 5'-TCACTACGTATCAGTGGTGGTGGTGGTGGTGGCCTGAACC-3 '(SEQ ID NO: 9) is used to amplify the gene encoding the PD-1-AP-His fusion protein, introducing restriction endonuclease sites Ngo MIV and Sna BI at the upstream and downstream of the gene, and the PCR conditions are 95 30 s at ℃, 30 s at 60 ℃, 60 s at 72 ℃, 35 cycles, extension at 72 ℃ for 10 min, the DNA polymerase is the product of TAKARA company.
[0051] After the PCR product was recovered by agarose gel (the recovery kit is a product of Omega bio-tek), after purification, restriction endonucleases Ngo MIV and Sna BI were added for digestion, and the digested product was purified by a DNA recovery reagent (Omega bio-tek The pr...
Embodiment 3
[0054] Construction and expression of anti-PD-1 human antibody eukaryotic expression vector
[0055] The amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 2, and the coding DNA sequence used in this example is SEQ ID NO: 1;
[0056] The amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 4, and the coding DNA sequence used in this example is SEQ ID NO: 3; Synthetic Technology Co., Ltd.)
[0057] PCR reaction is used to amplify the coding DNA sequences of the above-mentioned light chain variable region and heavy chain variable region, and introduce appropriate enzyme cutting sites at both ends of the gene of the antibody heavy chain variable region and light chain variable region, wherein Ngo MIV and SnaBI restriction sites were introduced at the 5' end and 3' end of the antibody heavy chain variable region gene respectively, and the forward and reverse primer sequences were: 5'-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCAT...
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