Chicken marrow-derived dendritic cell (DC) targeting peptide AH and application thereof
A technology of dendritic cells and targeting peptides, applied in the field of biomedicine, can solve the problems of poor binding effect of poultry-derived DCs, achieve the effects of reducing degradation rate and side effects, improving immune efficiency, and being easy to synthesize
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Embodiment 1
[0026] Example 1 Isolation, cultivation and identification of chicken bone marrow-derived dendritic cells
[0027] 1. Isolation and culture of chicken bone marrow-derived dendritic cells
[0028] Chickens about 30 days old were sacrificed and soaked in 75% ethanol for 10 minutes. The femur and tibia were taken out under aseptic conditions, and PBS containing 2% double antibody (pH 7.2) was sucked with a syringe, and the bone marrow was washed until the marrow cavity became white; For the washing solution, wash twice with PBS containing 2% double antibody, centrifuge at 1000r / min for 5-10min each time, and discard the supernatant; collect the precipitate and resuspend the precipitated cells with PBS, and then dilute the bone marrow cell suspension at a ratio of 1:1. Slowly add to the centrifuge tube containing Histopaque-1119, centrifuge at 2500r / min for 30min; collect the white misty cells at the interface in the middle, resuspend in PBS, centrifuge at 1500r / min for 10min, dis...
Embodiment 2
[0036] Example 2. Preliminary detection of phage display 12-peptide library subtractive screening for targeting peptides and their affinity for DC
[0037] 1. Phage display 12-peptide library subtractive screening targeting peptides
[0038] The screening process is divided into four rounds, as follows:
[0039] First round: place 2 x 10 11 The phage display 12 peptide library (PhD-12) library of pfu was added to the chicken bone marrow derived dendritic cells (5 × 10 5 / mL), and incubated at 4°C for 30min, centrifuged at 2000rpm for 3min, resuspended the cell pellet in PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween 20, and repeated washing 3 times; adding 1mL Buffer solution (0.2mol / L glycine-hydrochloric acid, pH=2.2, 1mg / mL bovine serum albumin), mix lightly at room temperature for 10 minutes, then add 50μl Tris-HCl (pH value 9.1) for neutralization; centrifuge, and the supernatant is The phage eluate was bound to the surface of the membrane, and the eluate...
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