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Ovary cancer specific tumor antigen peptide and preparation method thereof

A tumor antigen peptide, ovarian cancer technology, applied in the field of ovarian cancer specific tumor antigen peptide and its preparation, can solve the problem of low activation efficiency and the like

Active Publication Date: 2014-04-23
SHANGHAI YUYAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although naturally occurring APCs of naive T cells (e.g., dendritic cells, macrophages, B cells, or autologous tumor cells) that can be activated in vitro can also be used, since the MHC molecules of natural APCs contain many other peptides Antigenic epitope, the efficiency of its activation is low

Method used

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  • Ovary cancer specific tumor antigen peptide and preparation method thereof
  • Ovary cancer specific tumor antigen peptide and preparation method thereof
  • Ovary cancer specific tumor antigen peptide and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of Ovarian Cancer-Specific Tumor Antigen Peptides

[0041] 1. Cell culture

[0042] The cell line used for identification of antigenic peptides is HTB161A2. Cells were cultured adherently in RPMI supplemented medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Invitrogen). On the first day, the concentration of cells was adjusted to 0.5×10 6 cells / ml, generally speaking, when the cell concentration reaches 2×10 6 cells / ml (3-4 days) the cells will divide.

[0043] 2. Cell Collection

[0044] Pour off the culture medium from the T75 culture flask, wash the cells three times with pre-cooled 1×PBS buffer, and dilute the lysate with 3ml of cell lysis buffer [50mM Tris (pH8.0), 150mMNaCl, 1% CHAPS (Aldrich, Cat. No.226947), 5uM EDTA, 0.2% sodium azide, 17.4μg / ml PMSF (Calbiochem-Novabiochem), protease inhibitors (Roche, Cat.No.1697498)], wash and collect cells, and in 4 ℃ The reaction was carried out under rotation for 1 hour, the s...

Embodiment 2

[0055] Example 2: Detection of antigenic peptides

[0056] like figure 2 As shown, 1 μl of the above-mentioned retentate was added to the plate for protein analysis, and analyzed by Bioanalyzer. According to the control comparison, the left figure shows the chromatogram, and the right figure shows the electrophoresis figure, both of which show the existence of MHC I with a molecular weight of 44kD.

[0057] 5. Liquid chromatography tandem mass spectrometry (LC / MS / MS)

[0058] Peptide solutions isolated from HLA-A2-linked peptides were filtered and acidified with 1% trifluoroacetic acid (TFA). Inject 10 μl of EksigentnanoLC (Eksigent Technologies, Inc.) into each sample for desalting and capture as follows: capture column = LC filled C18Pepmap100, 5u, 100A, 300um id × 5mm; capture mobile phase = water, 2% methanol, 0.05% TFA ;Capture flow rate=5μl / min;Analytical column=Vydac;Everest C18,5u,300A,75um id×150mm;Analytical mobile phase: A=water,2%methanol,0.1%acetic acid;B=10%wa...

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Abstract

The invention relates to an ovary cancer specific tumor antigen peptide and preparation method thereof. The preparation method comprises the following steps: carrying out an adherent culture of cell HTB161A2 with a supplemental medium, and collecting a supernatant; collecting 0.5ml magnetic beads and washing with a coating buffer; suspending the magnetic beads again, filtering and eluting; collecting eluted substances in a centrifuge tube, and collecting a filtrate, and the filtrate is a solution of the antigen peptide. The method provided by the invention is a direct biochemical method, that is using an immuno-affinity purification method for purifying MHC / polypeptide compound molecules from tumor cells or tissues, and eluting the antigen peptides combined with MHC from the MHC / polypeptide compound molecules. These antigen peptides can be used for antibody treatment potentially, or producing ovary cancer specific CTLs, or preparing tumor vaccine; the invention also be developed for producing diagnosis kit.

Description

[technical field] [0001] The invention relates to a tumor antigen peptide and a preparation method thereof, in particular to an ovarian cancer-specific tumor antigen peptide and a preparation method thereof. [Background technique] [0002] Ovarian cancer is the most common cause of death in gynecological cancers in the United States. Early onset is minimal, nonspecific or even asymptomatic. Therefore, most patients can only be diagnosed at an advanced stage. Standard treatment consists of invasive surgery followed by chemotherapy. Ovarian cancer has been described histologically in many ways, however, more than 90% of malignancies are epithelial tumors. Approximately 22,430 new cases of ovarian cancer are diagnosed each year. Estimates indicate that 1 in 70 women will develop ovarian cancer in her lifetime. Ovarian cancer accounts for 3.3% of all new cancer cases. Researchers are investigating new approaches to treating ovarian cancer, including metalloproteinase inhib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06
Inventor 施炜星杨春霞房永生
Owner SHANGHAI YUYAN BIOTECH CO LTD
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