Medicine or cosmetic prepared from fibroblast extracellular vesicles
A technology of vesicles and monoclonal antibodies, applied in cosmetic preparations, animal cells, vertebrate cells, etc., can solve the problems of easy aging of fibroblasts, low activity of exosomes, low yield, etc., and achieve good market application value , good effect of anti-aging activity
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Embodiment 1P53
[0037] Example 1 Preparation of P53 monoclonal antibody
[0038] Recombinant human p53 protein (abcam, Cat. No. ab84768, sequence shown in SEQ ID NO: 1) was used as the immunogen. The recombinant protein was thoroughly mixed with an equal volume of Freund's complete adjuvant, and BALB / c mice were subcutaneously injected with 50 μg / mice of the recombinant protein. Four weeks after the initial immunization, multiple subcutaneous injections on the back were given to boost the immunization, and 4 weeks later, the immunization was boosted again, with 50 μg of the immunogen per animal each time. Seven to ten days after the third immunization, blood was collected from the tail vein to measure the serum antibody titer by ELISA. Four days before fusion, the mouse with the highest antibody titer was selected and boosted by intraperitoneal injection of 150 μg of unadjuvanted antigen.
[0039] Aseptically take spleen cells of booster immunized BALB / c mice, use 50% polyethylene glycol as...
Embodiment 22
[0041] Example 2 Affinity, isotype identification, sequence analysis and specificity identification of 2F6 monoclonal antibody
[0042] Using the AMC sensor, the purified 2F6 antibody was diluted with PBST to 10ug / ml, and the recombinant protein was diluted with PBST: 444.4nmol / ml, 222.2nmol / ml, 111.1nmol / ml, 55.6nmol / ml, 27.8nmol / ml, 0nmol / ml; operation process: equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM The pH 1.69 GLY solution and buffer 3 were used to regenerate the sensor and output data. The results are shown in Table 1. (KD stands for equilibrium dissociation constant or affinity)
[0043] Table 1 Affinity results of 2F6 monoclonal antibody
[0044] Antibody name KD(M) 2F6 monoclonal antibody 9.86E-09
[0045] It can be seen from the results in Table 1 that the 2F6 monoclonal antibody...
Embodiment 3
[0054] Example 3 Cultivation of fibroblasts
[0055] The fibroblast HSF was cultured in DMEM (containing 10% fetal bovine serum, 1% double antibody mixture) medium, placed in a 37°C, 5% CO2 incubator, and the cells grew to more than 80%, rinsed with PBS for 2- 3 times, add 1 mL of 0.25% trypsin to digest at room temperature for 30-60 s, tap the bottle wall to make the cells fall off, add 2 times the volume of fetal bovine serum-containing medium to terminate the digestion, pipette evenly with a gun tube, and centrifuge at 1000 r / min for 8 min , remove the supernatant, precipitate and add an appropriate amount of complete medium to make a single cell suspension, adjust the concentration, and use the obtained HSF in logarithmic growth phase in good condition for the experiment. HSF in logarithmic growth phase was inoculated into a 96-well culture plate at 1×105 cells / mL, and 90 μL was inoculated in each well, placed in a cell incubator at 37 °C, 5% CO2, and taken out after 24 h ...
PUM
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