Method of extracting and preparing deer DGF
A technology of epidermal growth factor and deer antler, which is applied in the field of medical or edible products of deer antler extract, and can solve problems such as restriction of dosage, waste of resources, protein denaturation such as EGF, etc.
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Embodiment 1
[0027] Example 1, Pretreatment of velvet antler
[0028] Take 0.5 kg of fresh velvet antler, use a cryostat to cut the velvet antler into slices with a thickness of 1 to 2 mm, and then add an appropriate amount (200 mL) of pre-cooled deionized water to crush with a homogenizer at 4°C, and proceed to homogenization. It ends when there are no visible tissue blocks in the slurry. Then, 200 mL of pre-cooled deionized water was added to the homogenate, and the mixture was mixed and ultrasonically broken under ice bath conditions. The resulting crushed product was centrifuged at 5000 revolutions per minute (rpm) for 20 minutes, and the supernatant was taken. Add 200 mL of pre-cooled deionized water to the centrifugal pellet, resuspend the pellet and centrifuge again at 5000 rpm for 20 minutes, and take the supernatant. Then, repeat the above resuspension and centrifugation process once, and take the supernatant. Combine the supernatants obtained from each centrifugal separation, put the...
Embodiment 2
[0029] Example 2. Chromatographic purification of velvet antler epidermal growth factor and preparation of lyophilized agent
[0030] Take 820 mL of the deer antler extract obtained in Example 1 and apply it to Q Sepharose TM Fast Flow column (column parameter 5.5cm×20cm) (purchased from Amersham), and then eluted. During elution, the impurity protein peak is eluted with 300mL 0.02mol / L Tris-HCl buffer (pH8.0) at a flow rate of 2mL / min, and then 450mL 0.02mol / L Tris containing 0.5mol / L NaCl -HCl buffer (pH 8.0) for elution, and use an ultraviolet detector to detect at a wavelength of 280 nm (the elution profile is shown in Figure 1). The product obtained by dialysis of each eluted fraction was detected by the detection method described in Example 3, and it was found that the peak referred to by the "target protein peak" in FIG. 1 was velvet antler epidermal growth factor. Therefore, collect the target protein peak, put it into a dialysis bag with a molecular weight cut-off of 3500...
Embodiment 3
[0031] Example 3. Detection of epidermal growth factor
[0032] According to the "Molecular Cloning Test Guide" (Science Press, 2002) and the biological activity detection method of Xiong Sheng et al. (Chinese Journal of Health Inspection, 2006, 16(10): 1153-1155, 1174), velvet epidermal growth factor was detected. The velvet antler epidermal growth factor solution obtained in Example 2 was subjected to SDS-PAGE (15% polyacrylamide separation gel, 4% layered gel, 10mA constant current electrophoresis), and stained with Coomassie brilliant blue after the electrophoresis. As shown in Figure 2, there is a single band of epidermal growth factor at a molecular weight of about 7kD, and its purity is 97.2% by image analysis and area normalization method. The proliferation of epidermal cells was used to detect the biological activity of EGF. The blank group added with buffer was used as a control. In the experimental group added with the velvet antler epidermal growth factor solution obta...
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