A kind of anti-cd3 antibody and its preparation method and application
An antibody, CDR2 technology, applied in the fields of biology and medicine, can solve the problems of inability to apply cell sorting and restricting the development of cell separation
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Embodiment 1
[0122] Example 1 Preparation and identification of mouse anti-human CD3 monoclonal antibody
[0123] Using the purchased CD3 protein as the immunogen, add complete Freund's adjuvant at a ratio of 1:1 for the first immunization, fully grind until completely emulsified, and inject 6-8 weeks old Balb / c mice subcutaneously at multiple points, 100 μl per point , a total of 10 points, followed by repeated injections of booster immunization every other week, a total of 5 times, adding incomplete Freund's adjuvant at a ratio of 1:1, fully grinding until completely emulsified, and then immunizing mice. The dose and method of immunization were the same as the initial immunization. Monitor serum antibody levels. Intrasplenic injection was used for the last immunization, and the dose was the same as before. The mice were sacrificed 3 days later, and the spleen was taken out. The spleen single-cell suspension was obtained by conventional methods, and the cell viability was detected to be ...
Embodiment 2
[0127]Example 2 Sequencing and amplification of the variable region of the mouse anti-human CD3 monoclonal antibody
[0128] 2.1 Determination of the heavy chain variable region sequence and light chain variable region sequence of the mouse anti-human CD3 monoclonal antibody
[0129] Since the sequence of the constant region downstream of the antibody gene is known, the appropriate positions of the constant region of the antibody heavy chain and light chain were selected, and three gene-specific primers (as shown in Table 1) were designed respectively. Amplification) method to obtain the variable region gene of anti-CD3 mouse monoclonal antibody m3E3 (amplification method such as figure 1 ).
[0130] Table 1 Synthetic primers (purchased from Shanghai Shenggong Company)
[0131] Primer name
Primer sequence
SEQ ID NO:
GSP1-H:
5'-TCATCCTTGCTGATGTCTACCACA-3'
1
GSP2-H:
5'-AGTAATGGTGAGCACATCCTTG-3'
2
GSP3-H:
5'-GGGAAGATGAAGAC...
Embodiment 3
[0158] Example 3 Construction of Vectors Containing Human Antibody IgG1 Constant Region Genes
[0159] Total RNA was extracted from human spleen cells with Triozl reagent (purchased from Invitrogen), Oligod(T) 15 After reverse transcription into cDNA, design specific primers (as shown in Table 3) for PCR to amplify the heavy chain γ gene huCH and light chain κ gene huCκ of human IgG1 respectively, and add restriction sites for the amplified sequences . The gene sizes were about 1003bp and 321bp, respectively, and were cloned into the pMD18-T vector, and positive clones were screened for sequencing analysis.
[0160] The correct clones verified by sequencing were designated as pMD18-huCH (contains the constant region of human IgG1 heavy chain) and pMD18-huCκ (contains the constant region of human IgG1 light chain).
[0161] table 3
[0162]
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