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Polypeptide fragment for preparing DC (Dendritic Cell) vaccine and DC vaccine

A polypeptide fragment and vaccine technology, applied in the field of biological vaccines, can solve the problems of high production cost and low safety of DC vaccines, and achieve the effects of low production cost, high safety and high product stability

Inactive Publication Date: 2019-04-05
英普乐孚生物技术(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a polypeptide fragment and a DC vaccine for preparing a DC vaccine, so as to solve the technical problems of low safety and high production cost of the DC vaccine in the prior art

Method used

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  • Polypeptide fragment for preparing DC (Dendritic Cell) vaccine and DC vaccine
  • Polypeptide fragment for preparing DC (Dendritic Cell) vaccine and DC vaccine
  • Polypeptide fragment for preparing DC (Dendritic Cell) vaccine and DC vaccine

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preparation example Construction

[0034] The preparation process of the DC vaccine of the present invention is as follows:

[0035] 1. Isolation and induction of DC cells:

[0036] Blood samples from breast cancer patients or leukocytes collected by apheresis machine are used to separate peripheral blood mononuclear cells with lymphocyte separation medium, and after the mononuclear cells are separated by adherent culture, they are treated with GM-CSF, IL-4, autologous plasma Lymphocyte culture medium to induce monocytes to differentiate into DC cells. The lymphocyte separation medium and lymphocyte culture medium used were directly purchased from the market.

[0037] 2. Peptide load:

[0038] After 5 days of induction culture, a polypeptide fragment derived from human lactoglobin was added to the culture system. The sequence is shown in Table 1. A polypeptide fragment of one sequence can be added alone, or a mixture of polypeptide fragments of several sequences can be added. The preferred concentration is 1...

Embodiment 1

[0044] Example 1 Design and Synthesis of Antigen Polypeptide Fragments

[0045] Obtain the amino acid sequence of human mammaglobin (also known as mammaglobin-A protein) from the NCBI database, and compare different polypeptide fragments with HLA-A0201 online (https: / / www-bimas.cit.nih.gov / molbio / hla_bind / ) Calculate the affinity and other data and score. The scoring results are shown in Table 2. The initial positions of the five predicted polypeptide fragments are concentrated in 2-4, and the scores are all high. Therefore, the polypeptide fragments of 2-10 and 4-12 are selected. Tested as a representative; in addition, the initial positions of 4 polypeptide fragments were concentrated at 80-84, and the scores were quite different, so the polypeptide fragments with higher scores of 83-92 and 80-89 were selected for detection; according to Table 2 A total of 9 polypeptides were selected for synthesis based on the above reasons, and their sequences are shown in Table 3. The po...

Embodiment 2

[0051] Example 2 Detecting the Binding Ability of the Polypeptide to HLA-A0201

[0052] 1) Reagents: IMDM medium, fetal bovine serum, cells expressing HLA-A0201T2, HLA-A02 antibody.

[0053] Polypeptide: positive control PI-9 (sequence is PYVSRLLGI, shown in SEQ ID NO.7), dissolved in DMSO, final concentration 20mg / mL; negative control GL-9 (sequence is GILGFVFTL, shown in SEQ ID NO.8) , dissolved in DMSO, with a final concentration of 20 mg / mL; the target polypeptide MAM01-06, whose sequence is shown in Table 2, was dissolved in DMSO, with a final concentration of 20 mg / mL. The dissolved peptides need to be subpackaged and stored at -80°C, and repeated freezing and thawing should be avoided during use.

[0054] 2) Method:

[0055] Cell culture: T2 cells (HLA-A0201), suspended in complete medium (IMDM+20% FBS), at 37°C, 5% CO 2 cultured under conditions.

[0056] Peptide loading: adjust the concentration of T2 cells to 1*10 with complete medium 6 / mL density, add differen...

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Abstract

The invention discloses a polypeptide fragment for preparing DC (Dendritic Cell) vaccine and the DC vaccine. The polypeptide fragment is selected from at least one of amino acid sequences as shown inSEQ ID NO. 1 to SEQ ID NO. 6. The DC vaccine contains polypeptide fragment loaded DC. Specifically, the DC vaccine is prepared from the polypeptide fragment loaded DC, human serum albumin and normal saline, wherein the mass concentration of the human serum albumin in the normal saline is 5 to 20 percent, and the density of the polypeptide fragment loaded DC is (0.5-2)*10<6> per mL. According to the polypeptide fragment for preparing the DC vaccine and the DC vaccine, disclosed by the invention, human mammaglobin is selected as an attack target spot, an effective DC polypeptide vaccine preparedfrom the polypeptide fragment obtained through computer design and experimental screening can be used for treating patients suffering from HER2 (Human Epidermal Growth Factor Receptor-2) positive / negative breast cancer, and preventing recurrence and metastasis. The DC vaccine is capable of reducing the production cost while increasing the vaccine safety.

Description

technical field [0001] The invention belongs to the technical field of biological vaccines, in particular to polypeptide fragments and DC vaccines for preparing DC vaccines. Background technique [0002] The incidence of breast cancer ranks first among female malignant tumors in the world, seriously endangering women's health; in China, the incidence of female breast cancer is also increasing year by year. With the development of early diagnosis and comprehensive treatment, the mortality rate of breast cancer has gradually decreased. Nevertheless, there are still many patients with metastasis and recurrence. [0003] In 1890, New York orthopedic surgeon William Coley found that patients with postoperative infection recovered better than those without infection when performing surgery to remove tumors, mainly because infection improved the body's immunity, which in turn improved immune cell recognition and The ability to kill tumor cells, this discovery puts forward a new i...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N5/0784A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C12N5/0639C07K14/47C12N2500/84C12N2501/22C12N2501/2304
Inventor 熊青卉武宁韩德平谢羽孙全霞
Owner 英普乐孚生物技术(上海)有限公司
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