Anti-human tim3 antibody or functional fragment thereof and application thereof
A functional, antibody technology, applied in the fields of biomedicine and antibody engineering, can solve problems such as incomplete activation of the immune system, achieve high sensitivity and specificity, broad development prospects, and improve accuracy.
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Embodiment 1
[0075] Example 1: Preparation of mouse anti-human Tim3 monoclonal antibody
[0076] (1) Animal immunity
[0077]The experimental animals were female Balb / c mice aged 6-8 weeks, which were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. After feeding for a week without any abnormality, they will be numbered. About 0.1 ml of blood was collected through the retro-orbital venous plexus. After standing at room temperature for 1 hour, overnight at 4°C, the supernatant serum was collected after centrifugation at 1500g, and stored at -20°C for future testing. For basic immunization, 100 μg / 100 μl of Tim3-His recombinant protein (ACRO Biosystems Company) was emulsified and mixed with an equal volume of Freund’s complete adjuvant (Sigma Company, product number F5881), and the immunization was performed by multi-point subcutaneous injection on the abdomen and back. The booster immunization was carried out at intervals of 3 weeks, and the same amount of Ti...
Embodiment 2
[0086] Example 2: Tim3 antibody sequence analysis
[0087] Amino acid sequence analysis of antibody variable regions Screening of phage antibody library by scFv confirmed four Tim3 monoclonal antibodies named MsT001, MsT065, MsT229 and MsT286.
[0088] Positive clones were sequenced to obtain light chain and heavy chain variable region sequences.
[0089] Through the analysis of IGBLAST software (https: / / www.ncbi.nlm.nih.gov / igblast / ), it was found that the scFv antibody belongs to VH category and Vκ type; ABodyBuilder software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ) in Kabat mode to further analyze the positions of the three CDR regions of each antibody VH (heavy chain variable region) and VL (light chain variable region).
[0090] The CDR sequences of the antibody heavy chain variable region are shown in Table 1 below.
[0091] Table 1
[0092]
[0093]
[0094] The amino acid sequence of the heavy chain variable region is as follows (t...
Embodiment 3
[0142] Example 3 Antibody Expression and Purification Identification
[0143] The same antibody light chain and heavy chain expression vectors were transiently transfected into 293 cells at the same time, and the culture supernatant was collected 6-7 days after transfection, and purified by protein A purification column for affinity purification to obtain purified antibodies. The molecular weight of the antibody was predicted by the protein molecular weight calculator, and the purity and molecular weight of the antibody were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
[0144] SDS-PAGE electrophoresis detection process: Take 5 μg of purified antibody, respectively, under non-reducing conditions (protein electrophoresis loading buffer without adding β-mercaptoethanol, samples are not boiled) and reducing conditions (protein electrophoresis loading buffer with β-mercaptoethanol, samples Boil for 5min), use 10% SDS-PAGE gel for electrophore...
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