The pharmaceutical small molecule compound of the vsig8 small molecule inhibitor and its medical use and pharmaceutical composition
A technology of small molecule inhibitors and compounds, applied in the field of biomedicine, can solve the problems of high price, complex preparation process of antibody drugs, inconvenient use, etc., and achieve the effect of promoting value-added
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Embodiment 1
[0040] Expression and preparation of human extracellular segment VSIG8-His fusion protein
[0041] Rabbit anti-human VSIG8 antibody (ab83852) was purchased from Abcam; rabbit secondary antibody (CW0156) was purchased from Biyuntian Biotechnology Co., Ltd.;
[0042] 1. Recovery culture of ExpiCHO-STM cells
[0043] Take the frozen ExpiCHO-STM cells out of liquid nitrogen, thaw them in a water bath at 38-39°C, and keep shaking them to make them melt quickly until only a small amount of ice cubes remain in the tube. Remove the cells from the water bath Take it out from the tube, spray it with 75% alcohol and open it in an ultra-clean bench, transfer the cell suspension (1mL) in the cryopreservation tube to a cell shaker flask containing 29mL Expi CHO expression medium (Gibco, A2910001) prepared in advance , place the cell shaker flask at 37°C, CO 2 The concentration is 8%, cultured in an incubator with a shaker rotation speed of 130 rpm.
[0044] Three days after recovery, whe...
Embodiment 2
[0077] Screening of Affinity Molecules of VSIG8 Protein by Competitive ELISA
[0078] Competitive ELISA method is used to screen the affinity molecules of VSIG8 protein, and the affinity of the screened objects to VSIG8 protein can be characterized through simple co-incubation, washing and color development steps. Mainly rely on the reaction of horseradish peroxidase and TMB chromogenic solution to produce a color change for detection. During the experiment, the VSIG8 protein was coated on the ELISA plate by physical adsorption, and the non-specific binding sites that were not saturated by the protein at the bottom of the plate were removed after washing and blocking steps, and then the test object and VSIG8-specific antibody mixture were added. Finally, secondary antibody and TMB were added for color development, and the absorbance at 450 nm was detected. If the detection object does not bind to the VSIG8 protein, the detection antibody will completely react with VSIG8, and th...
Embodiment 3
[0088] Microscale Thermophoresis (MST) assay was used to screen and identify the affinity molecules of VSIG8 protein. The ELISA method in Example 2 can characterize the affinity of the screening object for the VSIG8 protein through simple co-incubation, washing and color development steps. Molecules with better activity after screening by ELISA experiments will continue to be verified by MST experiments in the later stage to verify the affinity of the molecules with VSIG8 protein. The MST experiment refers to the directional movement of molecules in the microscopic temperature gradient field. Through the infrared excitation in the center of the capillary pipette and the cooling of the periphery, the temperature gradient difference in the microscopic area is generated in the capillary pipette, and the protein to be measured is compared with the small one. The molecular mixture solution is filled into the capillary pipette, and then the strength of the binding ability of the pro...
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