MAGE (Melanoma Antigen Gene)-4 anti-tumor CTL (Cytotoxic T Lymphocyte) epitope peptide and application thereof
A MAGE-4, 1.MAGE-4 technology, applied in the field of biochemistry, can solve the problem of not being able to achieve the effect of tumor suppression
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Embodiment 1
[0025] Example 1: Preparation of anti-tumor CTL epitope peptide P286 (Lys-Val-Leu-Glu-His-Val-Val-Arg-Val)
[0026] The Fmoc solid-phase synthesis method is used to extend one by one from the C-terminus to the N-terminus. Use fluorenylmethoxycarbonyl (Fmoc) to protect the α-amino group of amino acid, and the side chain protecting groups of various Fmoc protected amino acids are Arg (Pbf), His (trt), Glu (OtBu), Lys (Boc) (Pbf represents 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, trt represents trityl, OtBu represents tert-butyl ester, Boc represents tert-butoxycarbonyl). First, use N, N'-diisopropylcarbodiimide (DIC) as the condensing agent for the first amino acid at the C-terminal of the α-amino protected C-terminal, and add 1-hydroxybenzotriazepam azole (HOBt), Fmoc-Val-OH is connected to the wang resin; sequentially washed with DMF, anhydrous methanol, dichloromethane, DMF; then piperidine-DMF mixture (V 哌啶 :V DMF =1:4) to remove the Fmoc protecting group, follo...
Embodiment 2
[0028] Example 2: Preparation of anti-tumor CTL epitope peptide P286-1Y2L (Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Val)
[0029] The same synthesis method as in Example 1 was adopted, except that the second N-terminal amino acid Val was replaced by Leu, and Lys was replaced by Tyr.
[0030] The product was analyzed by mass spectrometry, see figure 2 , the result confirmed that the molecular weight was 1127.4, which was consistent with the theoretical value.
Embodiment 3
[0031] Example 3: Preparation of anti-tumor CTL epitope peptide P286-1Y2L9L (Tyr-Leu-Leu-Glu-His-Val-Val-Arg-Leu)
[0032] The same synthesis method as in Example 1 was adopted, except that the second amino acid Val at the N-terminus was replaced by Leu, the ninth amino acid Val at the N-terminus was replaced by Leu, and Lys was replaced by Tyr.
[0033] The product was analyzed by mass spectrometry, see image 3 , the results confirmed that the molecular weight was 1141.4, which was consistent with the theoretical value.
[0034] The CTL epitope peptide prepared above can be used to prepare a tumor therapeutic polypeptide vaccine, and its application experiment is as follows:
[0035] 1. Binding test of epitope peptide and HLA-A2.1 molecule
[0036] (1) Collect HLA-A2.1-positive T2 cells (ATCC company) with positive expression of HLA-A2.1 and lack of endogenous antigen processing ability by centrifugation at 800rpm, wash 3 times with phosphate buffered saline (PBS) at pH 7....
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