Preparation method of sika deer coronet micromolecular protein monomer
A technology of small molecular protein and sika antler, applied in the field of biological protein, can solve the problems of unsuitable small molecular protein and polypeptide, high cost, etc.
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Embodiment 1
[0035] A method for preparing small molecule protein monomers of sika antler discs, which is carried out according to the following steps:
[0036]Step 1: Extraction of total peptides of antler disc: Weigh 500g of antler disc powder, add 3000mL acetic acid-sodium acetate buffer solution with pH 3.6 fully pre-cooled in a refrigerator at 4°C, stir thoroughly with a magnetic stirrer, and then centrifuge the homogenate ( 4°C, 6000rpm, 25min), take the supernatant, and slowly add 95% ethanol that is fully pre-cooled in a 4°C refrigerator until the final concentration of the supernatant reaches 55%, place it in a 4°C refrigerator and stir once every 1h, after 4h Centrifuge (4°C, 6000rpm, 25min) to take the supernatant; use a vacuum rotary evaporator to evaporate the supernatant at 45°C until the volume of the concentrated solution is about 400mL; filter the concentrated solution and put it in a large petri dish, vacuum freeze Freeze-dry in a dryer to obtain the crude extract of tota...
Embodiment 2
[0060] HPLC purity test was performed on the target protein enrichment solution, and the instruments used were Shimadzu analytical HPLC instrument (LC-10ATVP type), ultraviolet spectrophotometric detector and binary gradient pump. ACE analytical reverse-phase HPLC column (ACE-221-2546), the specification of the chromatographic column is ACE 5 C18-3004.6×250mm analytical column. The gradient setting of elution is: acetonitrile concentration 0%, 0-5min; 0%-10%, 5-15min; 10%-60%, 15-45min; 60%-100%, 45-50min. The injection volume was 20 μL. The target peak appears when the B solution concentration is about 55.9%, and the corresponding time should be about 40 minutes. The result is as follows:
[0061] In the newly set gradient, the target protein appears at about 39.4min, which is in line with the expected judgment, and is determined by Figure 6 It can be seen that the purity of the target protein is high, meeting the sequencing requirements (the protein purity is above 89%)....
Embodiment 3
[0063] The target protein was analyzed by mass spectrometry, and the following results were obtained:
[0064] This part was assisted by the Institute of Basic Medical Sciences of Peking Union Medical College (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). Mascot is a protein search and identification software, and the search database is NCBI. The enzyme used in the enzymatic hydrolysis of the target protein enrichment solution is trypsin. After enzymatic hydrolysis, tandem mass spectrometry was used to identify the enzymatic fragments by MS / MS MS / MS. The identification results were compared with the NCBI protein database by Mascot software, and the matching proteins were found and analyzed. The specific query conditions are shown in the table 1.
[0065] Table 1 query conditions
[0066]
[0067] After enzymatic hydrolysis, use reverse high performance liquid to enrich the relative enzymatic fragments, and use tandem mass spectrometry to iden...
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