Anti-egfr humanized single domain antibody, fc fusion protein, heavy chain fab protein and application thereof
A single-domain antibody, fusion protein technology, applied in the direction of anti-animal/human immunoglobulin, anti-enzyme immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. Clinical application, poor tumor tissue permeability, and high production costs
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Embodiment 1
[0066] Example 1 Design and construction of anti-EGFR antigen-specific humanized single-domain antibody
[0067] (1) Original anti-EGFR single domain antibody
[0068] The amino acid sequences of two anti-EGFR single-domain antibodies derived from llama (Llama) are shown in SEQ ID NOs: 1-2 respectively.
[0069] (2) Humanization of anti-EGFR single domain antibody
[0070] The humanization method adopts the method of protein surface amino acid resurfacing (Resurfacing) and VHH humanization universal antigen binding complementary region grafting method (CDR grafting to a universal framework) to complete.
[0071] The humanization steps are as follows: homology modeling of anti-EGFR single-domain antibodies EG2 and EG10, and the modeling software is Modeller9. With reference to the amino acid sequences of the soluble human antibody DP-47 and the homologous sequence NBBcII10 antibody, the anti-EGFR single domain antibodies EG2 and EG10 were humanized. The humanization results ...
Embodiment 2
[0075] Example 2 Construction of anti-EGFR humanized single-domain antibody expression plasmid
[0076] Gene synthesis of the anti-EGFR single-domain antibodies EG2, EG10, EG2M1, EG2M2, EG10, EG10M1 and EG10M2 genes listed in Example 1, and then PCR products with restriction endonucleases BbsI and BamHI sites were obtained. Endonuclease BbsI and BamHI respectively treat PCR product and vector (pSJF2 vector) (kim Is. Biosic Biochem. 2002, 66(5): 1148-51, Chinese Patent Publication No. CN102321175A (ZL 201110280031)), after T 4 Ligase was used to ligate and recombine to obtain the plasmid sdAb-pSJF2 which can be expressed in E. coli with high efficiency, and the gene sequence was determined to confirm the correctness of its sequence.
[0077] 1) PCR amplification conditions for synthesizing VHH target gene, the composition of 50 μl PCR system:
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[0080] PCR reaction conditions:
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[0082] 5' primer—GAA GAAGAA GAC AA CAG GCC SVK GTG MAG CTG GWG ...
Embodiment 3
[0085] Example 3 Expression and purification of anti-EGFR humanized single domain antibody
[0086] The strain containing the plasmid sdAb-pSJF2 described in Example 2 was inoculated on an LB culture plate containing ampicillin at 37°C overnight. A single colony was selected and inoculated into 15 ml of LB medium containing ampicillin, and cultured overnight at 37°C on a shaker. Transplant 10 ml of the overnight culture into 1 L of 2YT medium containing ampicillin, incubate at 37°C with a shaker at 240 rpm, when the OD value reaches 0.4 to 0.6, add 0.5 to 1.0 mM IPTG, and continue to culture overnight. Centrifuge and collect bacteria. Bacteria were lysed with lysozyme, centrifuged, and the soluble single-domain antibody protein in the supernatant was collected. The protein with a purity of more than 95% was obtained by Ni+ ion affinity chromatography. figure 1 SDS-PAGE electrophoresis results of purified anti-EGFR single-domain antibody protein after nickel column purifica...
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