Tonoplast protein 1 immunogen polypeptide and application thereof
An immunogen, tonoplast technology, applied in animal/human proteins, specific peptides, medical preparations containing active ingredients, etc. The effect of inhibiting tumor growth, promoting immune response, and promoting T cell proliferation
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Embodiment 1
[0014] The tumor model was used to detect the in vivo activity of the vacuolar membrane protein 1 immunogen polypeptide.
[0015] 6-8 weeks old C57BL / 6 mice were randomly divided into 4 groups, half male and half male, 10 mice in each group. (1) Blank group; (2) Polypeptide low dose group; (3) Polypeptide medium dose group; (4) Polypeptide high dose group. An animal model of small cell lung cancer was established and immunized on the 3rd, 5th, and 7th day after tumor cell inoculation. The plan is: add the same volume of solvent to the blank group, and set 3 doses of peptides in the experimental group: 5, 10, 20 mg / Kg, and multiple injections around the tumor. After 21 days, observe the number of surviving mice and calculate the survival rate. The results showed that the polypeptide can effectively improve the survival rate of tumor-bearing mice, and the survival rate of the high-dose polypeptide group reached 85.92%.
Embodiment 2
[0017] The immunogenicity of the vacuolar membrane protein 1 immunogen polypeptide was evaluated by the method of detecting the antibody titers of the vacuolar membrane protein 1 immunogen in the immunized animals. There were 40 BALB / c white mice, and the mice were randomly divided into 4 groups with 10 mice in each group. (1) Blank group; (2) Immunogenic polypeptide low-dose group; (3) Immunogenic polypeptide medium-dose group; (4) Polypeptide high-dose group. Immunizations were performed on the 0th, 7, 14, 21, and 28 days of the experiment. The plan is: the blank group is added with the same volume of solvent, and the experimental group has 3 doses of peptides: 5, 10, and 20 mg / Kg, and multiple injections near the lymph nodes on the back of the mouse. On the 28th day, blood was collected from the orbital venous plexus, once a week, centrifuged at 12,000 rpm for 2 minutes, the plasma was separated and the supernatant was collected, and stored at -20°C for use. After dissolvi...
Embodiment 3
[0023] Proliferation of T lymphocytes: aseptically take mouse spleen, wash with 1640 medium for 3 times, grind with 5ml syringe core, filter with 200 mesh screen, make single cell suspension, centrifuge (1000r / min, 5min), discard Qing, Tris-NH 4 CL cracked the red blood cells, allowed to stand in an ice water bath for 3-5 min, centrifuged (1000 r / min, 5 min), discarded the supernatant, and washed the cells twice with sterile cold PBS. Finally, add 10% calf serum in RPMI 1640 culture medium (5ml) to suspend the cells, count the cells, and adjust the cell concentration to 5×10 6 Pieces / ml, cultured in 96-well culture plates.
[0024] The experiment set up a blank control group, a model group (conavalin A, purchased by sigma company), and a group of polypeptide doses (5, 10, 20 mg / ml). After adding 100μl / well of spleen lymphocyte suspension to each group, 100μl of 1640 culture medium was added to the blank control group, ConA was added to the model group (final concentration 5μg / ml)...
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