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Antigen epitope peptide and application thereof

An antigen epitope and antigen technology, applied in the field of genetic engineering and biomedicine, can solve the problems of low tumor killing efficiency, less secretion of cytokines, poor antigen presentation effect, etc., and achieve excellent killing effect, convenient synthesis, chemical properties and The effect of stabilizing thermodynamic properties

Active Publication Date: 2017-02-01
中美赛尔生物科技(广东)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing antigen epitope peptides HBsAg and / or HBcAg, HBVPre-S2 are loaded on the antigen presenting cells, the antigen presentation effect is poor, the secretion of cytokines is less, and the specificity cannot be effectively produced after co-culture with lymphocytes Cytotoxicity kills cells with low tumor killing efficiency

Method used

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  • Antigen epitope peptide and application thereof
  • Antigen epitope peptide and application thereof
  • Antigen epitope peptide and application thereof

Examples

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preparation example Construction

[0040] An embodiment provided by the present invention is prepared by artificial synthesis. The preparation method step of described artificial synthesis is:

[0041] Firstly, a synthetic epitope peptide (shown in SEQ ID NO: 1). The synthesis method is as follows: the HBV core epitope peptide and the HBV surface antigen epitope peptide are connected in series to obtain the epitope peptide (SEQ ID NO: 1). The HBV core epitope peptide is preferably HBcAg and / or HBsAg, and the present invention takes HBcAg18-27 (shown in SEQ ID NO: 2) as an example. The HBV surface antigen epitope peptide is preferably HBV Pre-S2, and the present invention takes HBV Pre-S2 44-53 (shown in SEQ ID NO: 3) as an example.

[0042] The present invention entrusts Nanjing GenScript Biotechnology Co., Ltd. to synthesize the HBV core epitope peptide HBcAg (SEQ ID NO: 2), the surface antigen epitope peptide HBV Pre-S2 (SEQ ID NO: 3) and The antigenic epitope peptide (SEQ ID NO: 1), and its sequence and m...

Embodiment 1

[0065] Example 1 Epitope Peptide Loaded DCs

[0066] 1) Preparation of epitope peptide

[0067] The present invention entrusts Nanjing GenScript Biotechnology Co., Ltd. to synthesize HBV core epitope peptide HBcAg (SEQ ID NO: 2), surface antigen epitope peptide HBV Pre-S2 (SEQ ID NO: 3) and antigen epitope peptide (SEQ ID NO: 3) NO: 1), and its sequence and molecular weight were identified by high-performance liquid chromatography and mass spectrometry, and the concentration was above 95%.

[0068] 2) Epitope peptide-loaded DCs

[0069] Depending on the solubility of the epitope peptide, it was dissolved in water or DMSO, and the concentration of the stock solution was 10 mg / ml. Sterilize by filtration with a 0.22 μm syringe filter, store at -20°C after aliquoting. When loading DCs with epitope peptides, the final concentration of epitope peptides was 40 μg / ml.

[0070] 3) Loading DCs with epitope peptides linked with FITC fluorescent groups

[0071] Experimental results ...

Embodiment 2D

[0072] The cultivation of embodiment 2DCs and CTL

[0073] (1) Take peripheral blood, add heparin for anticoagulation, then add ficoll (1.077g / ml) for density gradient centrifugation (20°C, 1500r / min, 15min), take interfacial cells, wash twice with PBS, 400r / min , 10min, wash 2 times. Cells were collected and placed in a 6-well plate filled with AIM-V medium and incubated in a carbon dioxide incubator for 1.5-2.0 h. Gently take out the 6-well plate, aspirate the supernatant and suspended cells into another culture dish for later use.

[0074] (2) Add culture medium and rhGM-CSF 800IU / ml, IL-4500IU / ml to the culture wells filled with adherent cells, culture in a carbon dioxide incubator, add cytokines once after 48h, and add TNF-α250IU on the fifth day of culture / ml, continue to culture, DC cells mature on the 7th day.

[0075] (3) Culture of T lymphocytes and induction of DC-CTL in vitro

[0076] The suspension cells in step (2) were cultured as T cells, and the cell conc...

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Abstract

The invention relates to the technical field of genetic engineering and biomedicines, in particular to an antigen epitope peptide and application thereof. The amino acid sequence of the antigen epitope peptide provided by the invention is shown as SEQ ID NO:1; the antigen epitope peptide is obtained by connecting HBcAg (or / and HBsAg) and surface antigen epitope peptide HBV Pre-S2 in series. Experimental results show that the mature of dendritic cells can be effectively induced, the antigen presentation capacity can be effectively improved and more cell factors can be secreted by adopting the antigen epitope peptide provided by the invention; after the antigen epitope peptide is co-cultured with lymphocytes, specific cytotoxicity can be produced to kill cells, so that the tumour killing efficiency is improved; the growth of a tumour can be effectively inhibited; an anti-tumour effect is achieved; a new choice is provided for clinical treatment of hepatitis B and related liver cancer thereof.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and biomedicine, in particular to an antigenic epitope peptide and its application. Background technique [0002] About 300 million people in the world are infected with hepatitis B virus (hepatitis B virus, HBV), and there are about 120 million people in China. As a high-endemic area of ​​hepatitis B, China accounts for nearly 50% of the total carrier rate of HBV surface antigen (hepatitis B virus surface antigen, HBsAg) in the world. 8% to 10% of people are HBsAg carriers. There are about 10 million patients suffering from chronic hepatitis B, and the incidence rate in recent years is about 0.158%. In addition, more than 80% of patients with chronic hepatitis are chronic hepatitis B patients, and the relative risk of suffering from primary hepatocytes (hepatocellular carcinoma, HCC) increases at least 300 times in people with chronic HBV infection. [0003] The total length of th...

Claims

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Application Information

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IPC IPC(8): C07K14/02C12N5/0783C12N5/0784A61K39/29A61K36/17A61P31/20
CPCA61K35/17A61K39/12A61K2039/5158C07K14/005C12N5/0638C12N5/0639C12N2506/11C12N2730/10122C12N2730/10134
Inventor 李陶林词雄盖宝华刘佳朱江
Owner 中美赛尔生物科技(广东)有限公司
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