Anti-VEGF single-domain antibody, humanized single-domain antibody, fusion protein constructed by single-domain antibody and IgG1-Fc, and application thereof
A single-domain antibody, protein technology, applied in the direction of anti-animal/human immunoglobulin, anti-growth factor immunoglobulin, antibody, etc., can solve the problems of complex structure, long production cycle and high cost of monoclonal antibody
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Embodiment 1
[0040] Example 1 Construction of anti-VEGF antigen-specific single-domain antibody library
[0041] 1) VEGF antigen immunization of alpacas
[0042] According to the conventional immunization method, the purchased VEGF antigen (Human VEGF Protein, Human, Recombinant, manufacturer Beijing Yiqiao Shenzhou, product number HPLC-11066-HNAH) was used to select adult healthy alpacas, and the antigen was injected subcutaneously on the back of the neck at multiple points. Add antigen and equal volume of Freund's adjuvant, immunize in 4-8 times, follow up and observe the absorption of mass at the injection site to confirm correct immunization. The immunization interval is 7-15 days. After the fourth immunization, serum is collected to determine the antigen immune titer. When the titer reaches 10,000 to 50,000 times or more (ELISA method), about 100ml of whole blood is collected, and lymphocytes are separated. Store at -80°C for later use.
[0043] 2) Isolation of alpaca peripheral blo...
Embodiment 2
[0057] Example 2 Acquisition of anti-VEGF single domain antibody
[0058] 1. Screening of VEGF-specific single-domain antibodies
[0059] The first round of VEGF protein concentration was 150 μg / ml, 150 μl / well, 1 microwell, and incubated at 4°C overnight.
[0060] The second round of VEGF protein concentration was 10-100 μg / ml, 150 μl / well, 5 microwells, and incubated at 4°C overnight.
[0061] The third round of VEGF protein concentration was 10-50 μg / ml, 150 μl / well, 5 microwells, and incubated at 4°C overnight.
[0062] Blocking: 1% CPBS, 300 μl / well, 37°C for 2 hours.
[0063] Table 1 Screening results of VEGF-specific single-domain antibodies
[0064] number of filters Total amount of added phage antibody library Eluent+Tris-HCl The number of single colonies Elution titer first round 5.6×10 11
300μl+200μl 10 50 / μl second round 5.25×10 11
150μl / well+350μl about 2500 2.5×10 4 / μl
third round 5.32×10 11
150μl / ...
Embodiment 3
[0067] Example 3 Construction of specific VEGF single domain antibody expression plasmid
[0068] The specific VEGF single-domain antibody gene obtained in Example 3 was amplified by PCR to obtain a PCR product with restriction endonucleases BbsI and BamHI sites, and the PCR product and vector were treated with restriction endonucleases BbsI and BamHI, respectively. (pSJF2 vector) (kim Is. Biosic Biochem. 2002, 66(5): 1148-51, Chinese Patent ZL 201110280031), approved by T 4 Ligase was used to ligate and recombine to obtain a plasmid sdAb-pSJF2 that could be highly expressed in E. coli, and the gene sequence was determined to confirm the correctness of its sequence.
[0069] 1) The PCR amplification conditions for obtaining the VHH target gene, the composition of the 50 μl PCR system:
[0070]
[0071] PCR reaction conditions:
[0072]
[0073] 5' Primer—GAA GAAGAA GAC AA CAG GCC SVK GTG MAG CTG GWG GAK TCT
[0074] 3' primer—gaagatctccggatccTGAGGAGACGGTGACCTGGGT
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