Anti-human Tim-1 antibody with neutralization activity
An antibody and variable region technology, applied in the fields of genetic engineering and cellular immunology
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Embodiment 1
[0067] Example 1 Screening Tim-1 Antibody
[0068] 1. Construction of phage antibody library
[0069]Extract mRNA from human lymphocytes, reverse transcribe it into cDNA, and use primers complementary to the conserved regions on both sides of the antibody variable region gene to amplify the heavy chain (VH) and The light chain (VL) gene is used to construct a library; the scFv single-chain antibody is expressed after being inserted into the phage vector, and the scFv is connected by an artificial linker between VH and VL.
[0070] 2. Screening of anti-human Tim-1 antibody
[0071] (1) The phages adhered to the human Tim-1-Fc protein, and the human Fc protein was used as a negative control, and the phages that bound to the Tim-1-Fc protein but not human Fc were retained.
[0072] (2) Repeated washing to remove non-specific binding, eluting and collecting the phage bound to Tim-1;
[0073] (3) Re-infect Escherichia coli to screen specific phage antibodies.
[0074] 3. PCR id...
Embodiment 2
[0080] Embodiment 2 Amplification of Monoclonal Antibody HF22
[0081] 1. Materials
[0082] Protein A Sepharose CL 4B column Protein column: product of Beijing Benyuan Zhengyang Biotechnology Co., Ltd.; pcDNA3.1 plasmid: Invitrogen; sources of other reagents and materials are the same as in Example 1.
[0083] 2. Methods and Results
[0084] (1) the monoclonal antibody HF22 light chain variable region gene obtained in Example 1, the light chain constant region gene is packed into pcDNA3.1 plasmid, and the carrier containing the monoclonal antibody HF22 light chain gene is obtained; the obtained embodiment 1 The heavy chain variable region gene of the monoclonal antibody HF22 and the heavy chain constant region gene are loaded into the pcDNA3.1 plasmid to obtain a vector containing the heavy chain gene of the monoclonal antibody HF22.
[0085] (2) Co-transfect the vector containing the light chain gene of the monoclonal antibody HF22 and the vector of the heavy chain gene in...
Embodiment 3
[0089] Example 3 Specific Detection of Tim-1 Protein of Monoclonal Antibody HF22
[0090] 1. Method
[0091] The specificity of monoclonal antibody HF22 was detected by ELISA.
[0092] (1) Coat 5 μg / ml human Tim-1 fusion protein in an ELISA plate, overnight at 4°C;
[0093] (2) Block unbound sites with skimmed milk powder, then wash 5 times with PBS buffer containing 0.2% Tween;
[0094] (3) Prepare different concentrations of anti-human Tim-1 antibody HF22 and control antibody, add to the ELISA strip in step (2), incubate at 37° C. for 1 hour, and wash 5 times with PBS buffer containing 0.2% Tween;
[0095] (4) Add HRP-labeled goat anti-human antibody, incubate at 37°C for 30 minutes, wash 5 times with PBS buffer containing 0.2% Tween; add TMB to develop color, and detect OD after 1mol / L sulfuric acid terminates 450 value.
[0096] 2. Results
[0097] The result is as figure 1 As shown, antibody HF22 can effectively bind Tim-1 antigen protein in a dose-dependent manner....
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