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Preparation method and application of anti-tumor vaccine antigen raw material

A vaccine antigen, anti-tumor technology, applied in anti-tumor drugs, biochemical equipment and methods, cancer antigen components, etc., can solve the problems of immune escape, complex extraction and preparation of tumor cell antigen composition, etc. The effect of simple immune escape, extraction and preparation process

Pending Publication Date: 2020-09-01
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide a preparation method and application of an anti-tumor vaccine antigen raw material, which is used in the manufacture of an anti-tumor vaccine, and solves the limitation that the extraction and preparation of tumor cell antigen compositions in the prior art are relatively complicated and prone to immune escape sexual problems

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  • Preparation method and application of anti-tumor vaccine antigen raw material
  • Preparation method and application of anti-tumor vaccine antigen raw material
  • Preparation method and application of anti-tumor vaccine antigen raw material

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Preparation and preservation method of anti-tumor vaccine antigen raw material

[0032] Cultivate tumor cells with 10% FBS (fetal bovine serum) medium in a 10mm×10mm petri dish until the cells in the dish reach about 5×10 6 At one hour, X-rays were used to irradiate with a dose of 20GY. The cells in the culture dish were collected on the third day after the irradiation. After the cells were broken, they were centrifuged by gradient centrifugation. Centrifuge at 14000g for 60min, discard the supernatant, and the resulting precipitate is the tumor vaccine antigen raw material.

[0033] The obtained precipitate was washed twice with normal saline, resuspended in 1ml PBS (phosphate buffered saline) solution, and stored at 4°C. After centrifuging 100 μl of the liquid, an appropriate amount of protein lysate was added, fully lysed at 0°C for 30 minutes, and then centrifuged at 12,000 g for 30 minutes. The supernatant was taken and added to BCA quantitative solutio...

Embodiment 2

[0034] Example 2: Expression experiment of MHC I and CRT on the surface of anti-tumor vaccine antigen raw materials.

[0035] Experimental steps:

[0036] (1) Mouse-derived lung adenocarcinoma cells Lewis received X-rays 8Gy, 20Gy, 8*3Gy;

[0037] (2) Add trypsin to digest each group of cells into a single cell suspension and collect them in EP tubes;

[0038] (3) Centrifuge in a low-speed centrifuge at a speed of 1000rpm / 5min, discard the supernatant after centrifugation, add 1mL of PBS buffer, mix well and centrifuge again at a speed of 1000rpm / 5min for 3 times, discard the supernatant, and use 200μL of PBS buffer Resuspend, add to the flow tube and mark it;

[0039](4) Add 1 μ antibody (CRT:FITC, MHC-I:FITC) to each flow tube and incubate at 4°C in the dark for 20 minutes;

[0040] (5) Add 1mL of PBS buffer, mix well, centrifuge 3 times at 1000rpm / 5min, discard the supernatant, add 200μ PBS to resuspend, and test on the machine.

[0041] Experimental conclusion: 20Gy ra...

Embodiment 3

[0042] Example 3: Experiment on the stimulating effect of anti-tumor vaccine antigen raw materials on bone marrow-derived dendritic cells.

[0043] Experimental steps:

[0044] (1) Mouse-derived lung adenocarcinoma cells, Lewis, were plated on a 6-well plate and irradiated with 8Gy, 20Gy, and 8*3Gy;

[0045] (2) Extract the cells of the control group and each radiotherapy group, use ultrasonic means to break the cells, and then centrifuge at a speed of 2000rpm / 10min, discard the precipitate and take the supernatant, and centrifuge at 10000rpm for 60min to obtain the precipitate, which is the tumor cell membrane;

[0046] (3) extract mouse bone marrow-derived mononuclear cells and differentiate them with GM-CSF to induce the formation of DC cells;

[0047] (4) Add the tumor cell membrane extracted from every 1 million tumor cells to 500,000 DC cells for co-culture for 6 hours, and then collect the DC cells into a clean EP tube;

[0048] (5) Centrifuge in a low-speed centrifug...

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Abstract

The invention provides a preparation method and an application of an anti-tumor vaccine antigen raw material. The method comprises the following steps: (1) obtaining in-vitro tumor tissues and separating and extracting primary tumor cells; (2) collecting the primary tumor cells obtained in the step (1) and then carrying out radiotherapy treatment, and (3) carrying out crushing and gradient centrifugation on the cells subjected to radiotherapy obtained in the step (2), and collecting tumor cell membranes carrying new antigens generated by radiotherapy induction so as to obtain the anti-tumor vaccine antigen raw material. The anti-tumor vaccine antigen raw material provided by the invention can be used for preparing an anti-tumor vaccine, and an antigen generated after radiotherapy is used as a vaccine antigen, so that the treatment effect of the tumor vaccine can be improved, and the limitation of a conventional tumor vaccine is broken through.

Description

Technical field: [0001] The invention relates to the technical field of anti-tumor vaccine preparation, in particular to a preparation method and application of an anti-tumor vaccine antigen raw material. Background technique: [0002] In the field of immunotherapy, vaccine-based therapy is an effective treatment because vaccine therapy can induce and expand immune cells against antigens. Bacterial and viral vaccines have saved tens of millions of human lives . However, the development and therapeutic effects of tumor vaccines have not met the ideal expectations, because tumor cells in different individuals have different antigen protein expression profiles, and immune cells in different individuals also have different immune cell response profiles, so In the process of vaccine design, the selection of antigens is difficult to unify, and tumor cells have the ability to escape from the immune system. Once individual tumor cells down-regulate or silence vaccine-associated ant...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P35/00C12N13/00C12N5/09
CPCA61K39/0011A61P35/00C12N13/00C12N5/0693
Inventor 金红林杨坤禹贺乾元庹展万超
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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