GP73 inhibitor and application thereof
A technology of inhibitors and inhibitors, applied in antiviral agents, antibodies, digestive system, etc., to achieve the effect of reducing the incidence of liver cancer
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Embodiment 2
[0037] Example 2 The high secretion of GP73 can cause high cell proliferation
[0038] In order to prove that the high secretion of GP73 can cause the high proliferation of cells, we put 2×10 6 The GP73 high-expressing cell lines HepG2GP73 and HepG2v were cultured in DMEM medium at 37°C, 5% CO 2 After culturing in a constant temperature incubator for 48 hours, the conditioned medium was collected. HepG2, MEF, MHCC97H, MHCC97HshGP73, PC3 cells and normal mouse fibroblast cell line (MEF) were respectively cultured with conditioned medium, and cell proliferation was detected. The details are as follows: 3000-5000 cells were seeded in a 96-well plate at 37°C, 5% CO 2 After incubating in a constant temperature incubator for one day, the normal culture medium was removed, and 200 μL of the conditioned medium was added, and the control was not replaced. MTT was performed every 24 hours to detect cell proliferation. Steps: Add 200 μL of fresh medium and 20 μL of MTT reagent [3(4...
Embodiment 3
[0041] Example 3 Hepatocellular carcinoma can be treated by GP73 inhibitor-shGP73
[0042] The level of cell proliferation after inhibiting GP73 was detected at the cell level, and the possibility of treating hepatocellular carcinoma by GP73 inhibitor was explored. We first knocked down GP73 in the liver cancer cell lines HepG2.2.15 and MHCC97H by the RNAinterference method, and found that shGP73 could inhibit the proliferation, plate colony formation and subcutaneous tumorigenesis of liver cancer cells ( image 3 I).
[0043] image 3 In A and D, shGP73 was expressed to knock down GP73 in the liver cancer cell lines HepG2.2.15 (A) and MHCC97H (D), and the expression level of GP73 was detected by western blot with empty load as the control. B and E, cell proliferation curves of HepG2.2.15shGP73 (B) and MHCC97HshGP73 (E). C and F, plate clones of HepG2.2.15shGP73 (C) and MHCC97HshGP73 (F). G, statistics of plate cloning experiments; H and I, tumorigenesis (left) and survi...
Embodiment 4
[0044] Example 4 Preparation of chimeric antibodies that can specifically recognize GP73
[0045]GP73 monoclonal antibody was entrusted to Jingtiancheng Biotechnology Co., Ltd. to prepare. Monoclonal antibody was used to immunize Balb / c mice with the human GP73 C-terminal peptide expressed by prokaryotes. From the splenocytes and myeloma cells of the immunized mice, the resulting hybridomas were plated on a 96-well plate, and tested by ELISA and Western blotting ( Figure 4 A-B) Positive clones were screened by detecting their ability to recognize recombinant GP73 molecules. Positive clones were subcloned and amplified, injected into mice to produce ascites, and antibodies were obtained after purification. The specific recognition of the antibody to tumor cells with high expression of GP73 was identified by flow cytometry analysis ( Figure 4 C), specifically as follows: 10 6 Cells were incubated with 8A10GP73 monoclonal antibody (40 μg / ml), and then PE-labeled mouse IgG...
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