Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar

[0020] Example 1: Expression, purification and biological activity determination of CBD-IL-10

[0021] (1) Acquisition of CBD-IL-10 gene

[0022] 1. Extraction of total RNA

[0023] Mononuclear cells were separated from human peripheral blood by lymphocyte separation medium, washed twice with PBS, resuspended in 10% RPMI 1640 culture medium, added ConA with a final concentration of 10g / L, placed in a 5% CO2 incubator, and incubated at 37°C for 48h. Collect cells. According to the method of RNA extraction kit, total RNA was extracted and stored at -80°C for later use.

[0024] 2. Extraction of total RNA and amplification of IL-10 cDNA

[0025] Use the RNA extraction and inversion kit from Takara Company to extract total RNA according to the instructions, and perform RT-PCR reaction: total RNA 500ng, 5× reverse transcription Buffer 2μl, oligo dT 0.5μl, 6mer 0.5μl, AMV reverse transcriptase 0.5μl , DEPC water was added to 10 μl. Incubate at 37°C for 30 minutes, then incubate...

[0057]Example 2: CBD-IL-10 inhibits scar hyperplasia test

[0058] (1) Hypertrophic scar tissue

[0059] The hypertrophic scar tissue of the forearm (proven by pathology) 3 months after the burn was healed was red, hard and about 4-7 mm higher than the normal skin. Thirty BALA / c-nu male mice, aged 6-8 weeks and weighing 18-20 g, were used.

[0060] (2) Preparation of animal model of post-burn hypertrophic scar

[0061] The adipose tissue under the hypertrophic scar was removed, and the scar was cut into 6mm×5mm×3mm tissue pieces, placed in pre-cooled DMEM (containing 100 U / ml of penicillin and streptomycin double antibodies) under sterile conditions A small incision was made in the skin of the scapula of nude mice, and a small piece of scar tissue was transplanted under the skin without suturing. Complete 30 animal models within 3 hours. Observe whether the animal model has infection or rejection, and select a stable scar model as the treatment animal. The scar model was ...

[0070] Example 3: Collagen Specific Binding Identification

[0071] Identification by ELISA: after neutralization of the collagen soluble by sarcosine, add it to a 0.1 mg / well 96-well plate, overnight at 4°C, wash 3 times with PBST, add 200ul of 0.25% BSA to each well to block for 2 hours at room temperature. The same TBST was washed 3 times, and 0.157-10 uM CBD-IL-10 was added to the corresponding wells in 3 parallel wells. The control group was added with the same concentration of IL-10, incubated at 37°C for 1 hour, and washed 3 times with PBS. Add 50 ul mouse anti-IL-10 monoclonal antibody (diluted 1:1000) and incubate at room temperature for 1 hour, wash 3 times as above, add 100 ul alkaline phosphate-labeled goat anti-mouse antibody (diluted 1:10000), incubate at room temperature for 1 hour , wash 3 times as above. Add 100 ul of 2 mg / ml nitrobenzene phosphate ( p -NPP) at room temperature for 10 minutes, adding 100 ul of 0.2M NaOH to each well to terminate the reaction...