Pilose antler basic fibroblast growth factor (DEER FGF) extraction and preparation thereof
A technology of growth factor and growth factor activity, applied in the field of deer antler extract rich in basic fibroblast growth factor, can solve the problem of not providing elution peak map
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Embodiment 1
[0028] Example 1, pretreatment of deer antler
[0029] Take 0.5kg of fresh velvet antler, cut the velvet antler into slices with a thickness of 1-2mm with a cryostat, and then add an appropriate amount (200mL) of pre-cooled deionized water to pulverize it with a homogenizer at 4°C, and the homogenization operation is carried out until uniform. End when no tissue mass is visible in the pulp. Then, 200 mL of pre-cooled deionized water was added to the homogenate, and after mixing, ultrasonication was performed under ice bath conditions. The resulting crushed product was centrifuged at 5,000 revolutions per minute (rpm) for 20 minutes, and the supernatant was collected. 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 process of resuspension and centrifugation once, and take the supernatant. The supernatants obtained by each centrifugal sepa...
Embodiment 2
[0030] Embodiment 2, the chromatographic purification of deer antler basic fibroblast growth factor and the preparation of freeze-dried agent
[0031] 60 mL of the deer antler extract obtained in Example 1 was loaded on a Sephadex G75 gel column (2.6 cm × 110 cm) (purchased from Amersham Company), and then eluted with distilled water at a flow rate of 80 ml / h, and was eluted with an ultraviolet detector. Detection was performed at a wavelength of 280 nm (the elution profile is shown in Figure 1). The products obtained by dialysis of each eluted fraction were detected by the detection method as described in Example 3, and it was found that the peak indicated by "target protein peak" in Figure 1 was deer antler basic fibroblast growth factor. Therefore, the target protein peak was collected, thereby obtaining 55 mL of purified velvet antler basic fibroblast growth factor solution. The obtained deer antler basic fibroblast growth factor solution was put into a dialysis bag and c...
Embodiment 3
[0032] Example 3, detection of basic fibroblast growth factor
[0033] Deer antler basic fibroblast growth factor was detected according to "Guidelines for Molecular Cloning Test" (Science Press, 2002) and MTT colorimetry of Chen Qiongyu et al. (Chinese Journal of Pathophysiology, 2006, 22(2): 247-250). The deer antler basic fibroblast growth factor solution obtained in Example 2 was subjected to isoelectric focusing electrophoresis (using 7.5% polyacrylamide as the supporting medium, using an amphoteric dielectric with pH 3.5 to 10, and focusing at 150V for 4 hours), and after the electrophoresis, the Coomassie brilliant blue staining, the electrophoresis gel photo is shown in Figure 2, there is a single band of basic fibroblast growth factor at about pH 9.5, and the image analysis by the area normalization method shows that its purity reaches 96.8%. Use the proliferation of Schwann cells to detect the biological activity of bFGF: with the blank group added with buffer as a c...
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