Anti-s100p protein monoclonal antibody and its use
A monoclonal antibody, protein technology, applied in the direction of anti-animal/human immunoglobulin, instruments, biochemical equipment and methods, etc., to achieve the effect of improving specificity and reliability
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Embodiment 1
[0037] Embodiment 1, the construction of S100P recombinant expression plasmid
[0038] Using Origene's S100P cDNA cloning plasmid SC116407 as a template, two primers were designed and introduced restriction sites SgfI and MluI respectively, and cloned into the expression vector pCMV6-Entry to establish the S100P recombinant expression plasmid RC201533. The cloning site is designed as figure 1 shown.
Embodiment 2
[0039] Embodiment 2, expression and purification of S100P recombinant protein
[0040]1. Transfect HEK293T cells: transfer HEK293T cells to culture dishes at a ratio of 1:3 and continue to culture; take 7.5mL DMEM (without serum and antibiotics) into a 50mL tube, add 300μL PEI MegaTran1.0 and mix well; add 75μg S100P recombinant plasmid DNA to In the mixed solution, mix well and let stand for 30 minutes; respectively take 515 μL into each culture dish and store at 37°C 5% CO 2 cultured in an incubator. 24 hours after transfection, add 25 μL of 2M sodium butyrate to a final concentration of 5 mM per dish of cells.
[0041] 2. Cell lysis: 48 hours after transfection, the cells were lysed. Aspirate the culture medium, add 1mL PBS for rinsing, and aspirate the PBS. Add 1 mL of lysis buffer and add protease inhibitors PI and PMSF just before use. Place in an ice box and vibrate on a shaker, collect the lysate from all culture dishes, centrifuge at 4 degrees, and collect the sup...
Embodiment 3
[0045] Example 3, Preparation and Screening of S100P Monoclonal Antibody
[0046] Purified recombinantly produced S100P protein fragments were used to immunize B6 / C57 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) according to standard methods. The specific method is as follows:
[0047] 1. Animal immunization: The purified S100P antigen was emulsified with complete Freund's adjuvant, and immunized 6-8 week-old BALB / c mice by subcutaneous or intraperitoneal injection. For the second immunization, it was emulsified with incomplete Freund's adjuvant, and the immunization dose was 50 μg per mouse. After the two immunizations, the tail blood was taken to measure the serum titer by ELISA gradient dilution; according to the results, it was determined whether to boost the immunization, and the mouse with the highest antibody titer was selected for cell fusion.
[0048] 2. Cell fusion: myeloma cells are sp2 / 0 derived from BALB / c, and they are in the logarithmi...
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