Optimized targeted MD-2 efficient anti-inflammatory polypeptide
A MD-2, targeted technology, applied in the field of biomedicine, to achieve high affinity, strong anti-inflammatory effect, and reduce the effect of LPS stimulating macrophages to synthesize and release inflammatory mediators
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[0036] Example 1. Construction of phage mutant peptide library
[0037] 1.1 Basic primers and mutant primers
[0038] Basic primer:
[0039] 96Etp: CATGCCCGGGTACCTTTCTATTCTCACTCT (for construction and colony PCR identification)
[0040] T6:TTTCGGCCGAACCTCCACCATGATTATCCGGCACCGTCTTAGAGTGAGAATAGAAAGGT
[0041] T11:TTTCGGCCGAACCTCCACCATTCCTAGTCAACGGAGAATCAGAGTGAGAATAGAAAGGT
[0042] 96gIIIsp: CCCTCATAGTTAGCGTAACG (for sequencing and colony PCR identification)
[0043] Synthetic strategy of mutant primers:
[0044] The T6 and T11 binding peptides are screened against the huMD2 mimic short peptide (FSKGKYKCV). The sequence is located at one end of the β-sheet chain of the huMD2 protein and the adjacent random coiled region, and it is basically linearly distributed. In order to obtain peptides with higher binding capacity, based on the T6 and T11 sequences, randomly mutate the adjacent 3-5 amino acids each time, retaining the original 2-4 amino acids, and at the same time at the amino and carbox...
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[0081] Example 2. Screening of phage peptide library
[0082] 2.1 Enrichment of specific phage clones that have binding activity to MD2 protein
[0083] Immune tube coating: Dilute antigen huMD210μg to 1ml with sterile TBS buffer, and coat the immune tube (Nunc, MaxiSorp) overnight at 4°C. Immune tube blocking: Use 5ml of 1% BSA blocking solution to seal the immune tube and incubate at 37°C for 2h. Washing: Discard the blocking solution, and wash the immunotube 3 times with TBST (T: 0.1% Tween-20). Phage binding: Add 3ml of phage sample (containing 0.5% BSA and 0.1% Tween-20), incubate at 37°C for 2 hours, and mix once every 30 minutes. Washing: Wash the immune tube 6 times with TBST (T: 0.1% Tween-20) to wash away unbound phage. Elution: Elute the bound phage with Glycine-HCl (pH 2.1), 1 mL / time, 5 min each time, and then add 160 μl of Tris neutralization solution. Repeat the elution once and then combine. Infection: From the eluted phage sample, take 10μl for gradient diluti...
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[0104] Example 3. Cytokine production inhibition test
[0105] 3.1 Cell culture
[0106] RAW264.7 was purchased from ATCC, and human pre-monocytes (THP-1) were donated by the Fourth Research Laboratory, Institute of Field Surgery, Third Military Medical University. RAW264.7 cells and THP-1 cells are cultured in RPMI1640 complete medium containing 10% fetal bovine serum. THP-1 cells need to be stimulated by phorbol ethyl ester (Phorbolmyristate acetate, PMA) to differentiate into mononuclear macrophages . After THP-1 is cultured and counted, add PMA100ng / ml and co-culture for 24h, adjust the cell count to 1×10 5 Pcs / hole. RAW264.7 directly adjusts the cell count to 1×10 5 Pcs / hole.
[0107] 3.2 Determination of the inhibitory effect of 5 clones on the cytokine production of RAW264.7 and THP-1
[0108] After RAW264.7 and PMA-induced THP-1 were washed with PBS for 3 times, the cell concentration was adjusted to 1×10 with 1640 culture medium containing 10% fetal bovine serum 5 Pcs / hole...
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