Method for presenting and activating DC cells in vitro and its application
A cell and fusion protein technology, applied in the field of in vitro presentation and activation of DC cells, can solve the problems of unknown tumor antigens, immature treatment methods, and difficulty in specific cell-targeted immunotherapy, achieving high safety and mild rejection. , to ensure the effect of controllability
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Embodiment 1
[0049] In this embodiment, the HBVc-MAGE-3 fusion protein is constructed by genetic recombination, and the molecular weight of the HBVc-MAGE-3 fusion protein is 22.4kD; the preparation steps of the HBVc-MAGE-3 fusion protein include: constructing a plasmid to express HBVc-MAGE through Escherichia coli -3 fusion protein, separation and purification of fusion protein and desalting by ultrafiltration.
[0050] HBVc-MAGE-3 fusion protein characteristics: HBVc virus-like particles (HBVcVLPs) can be used as vaccine adjuvants. HBVc virus-like particles have their characteristic immunogenicity, so they can be used as effective carriers for other proteins or peptides. The B cell antigen epitope or the main immunogenic region (mostimmunogenic region, MIR) of HBVc protein is located at the top of the spike-like protein coated on the HBVc antigen shell, and is arranged regularly, which is conducive to its interaction with the B cell receptor (BCR). coupling. The MAGE-3 peptide was recom...
Embodiment 2
[0103] In the present embodiment, the rMVA-MAGE-3 vaccine is constructed by gene recombination technology, and the preparation method of the rMVA-MAGE-3 vaccine comprises:
[0104] Construct shuttle plasmid: A549 cell RNA reverse transcription obtains MAGEA3 gene cDNA; MAGEA3 gene cDNA is cloned in pIIIdHR-P7.5 plasmid;
[0105] Recombinant MVA virus construction: CEF cells or BHK-21 cell monolayers grow to 70-90% coverage, sequentially infected with MVA, transfected with shuttle plasmids, and cultured to obtain monolayer cells; the monolayer cells are freeze-thawed and broken to obtain rMVA solution ; the rMVA solution was inoculated into RK-13 cell cultures to obtain rMVA infection RK-13 aggregation points, and the aggregation points were picked for screening and purification to obtain the required rMVA; the wtMVA in the rMVA was removed, and the rMVA was cultivated in CEF or BHK- In the 21-cell monolayer, rMVA without the selection gene K1L was screened, rMVA-FS was pur...
Embodiment 3
[0131] In this example, the specific MAGE-3 antigen peptide was directly synthesized.
[0132] Specific MAGE-3 antigen peptide characteristics: Predict the position of fragments that may have antigen-specificity in the polypeptide chain of MAGE-3 protein through bioinformatics calculations, synthesize corresponding fragment polypeptide chains (about 10-15aa) by chemical synthesis, and pass through cells And animal experiments to verify the antigen specificity of the polypeptide chain. A mixture of polypeptide chains with good antigen specificity was selected to impact DC cells, so that DC cells presented more MAGE-3 antigens. The amino acid sequence of the specific MAGE-3 antigen peptide is: FFPVIFSKASSLQL, EVDPIGHLY.
[0133] 1. The synthesis method of specific MAGE-3 antigen peptide is as follows:
[0134] 1. Anchoring: anchor the first amino acid on the solid phase resin;
[0135] 2. Deprotection: the protected amino acid uses an alkaline solvent to remove the protective...
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