A kind of hybridoma cell and the monoclonal antibody it produces and application
A hybridoma cell, monoclonal antibody technology, applied in the field of monoclonal antibodies, to achieve the effect of inhibiting thrombosis and inhibiting coagulation reaction
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Embodiment 1
[0041] Example 1: Preparation of Monoclonal Antibody Hybridoma Cells
[0042] 1. Materials
[0043] Antigen: protein with the amino acid sequence shown in SEQ ID NO:1, fully synthesized by the reagent company;
[0044] Test animals: 8-10 weeks old, about 17g BALB / C mice;
[0045] Reagents: Freund's complete adjuvant and incomplete adjuvant (purchased from Promega);
[0046] Myeloma cells: SP2 / 0 cells (provided by China Center for Type Culture Collection);
[0047] Medium: HAT selective medium (purchased from Sigma);
[0048] 2. Method
[0049] Get 100 μg of the protein with the amino acid sequence shown in SEQ ID NO:1 and add equal mass Freund's complete adjuvant to carry out the first subcutaneous injection to BALB / C mice, take the protein 300 μg with the amino acid sequence shown in SEQ ID NO:1 after 21 days, add The same mass of Freund's incomplete adjuvant was used to subcutaneously inject BALB / C mice for the second time, and after 10 days, the tail blood was collecte...
Embodiment 2
[0051] Example 2: Secretion produces DuIn antibody
[0052] After immunizing BALB / C mice with incomplete adjuvant for 1 to 2 weeks, intraperitoneally inject 1×10 6 A hybridoma cell with a preservation number of CGMCC NO.9153, which produces ascites 7 to 10 days after inoculation of the hybridoma cell;
[0053] Extract the ascites from the peritoneal cavity of the BALB / C mouse, put it in a 15ml centrifuge tube, centrifuge at 2000rpm for 10min to precipitate blood cells and other impurities, transfer the supernatant into a new centrifuge tube, store it in a -80°C refrigerator, and purify Before, use 0.1M PBS solution to dilute ascites at a ratio of 1:3;
[0054] Take 0.5ml Protein G and load it into the adsorption column, add 20 times the column volume of 0.1M PBS buffer to equilibrate the column for sample loading;
[0055] Take the diluted ascites, add it to the purification column, 2-3ml each time, and circulate the ascites through the column for 3 times;
[0056] After th...
Embodiment 3
[0059] Embodiment 3: DuIn antibody specific detection
[0060] Take ERp57fl / fl (control mouse, the gene encoding the amino acid sequence protein shown in SEQ ID NO: 1) and PF4-Cre / ERp57fl / fl (platelet-specific knockout of the amino acid sequence shown in SEQ ID NO: 1) Protein coding gene mouse) platelet lysate 200 μg and ERp57 recombinant protein 50ng were subjected to polyacrylamide gel electrophoresis, and then the protein was transferred to PVDF membrane and blocked with 5% skimmed milk solution at room temperature for 1 hour. Use TBS / T solution to dilute the anti-DuIn antibody to 1 μg / ml, put the blocked PVDF membrane into the antibody diluent, and incubate at room temperature for 2 hours. After the incubation, wash with TBS / T solution 3 times, 10 minutes each time, then add the membrane to the fluorescently labeled secondary antibody dilution solution, and incubate at room temperature for 1 hour. After 1 hour, wash the membrane 3 times with TBS / T solution, use Odyssey in...
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