Method for extracting thymosin, medium molecular thymus protein and DNA from thymus tissue
A technology of molecular weight and thymosin, applied in the field of thymus extract, can solve the problems of environmental pollution and waste of resources, and achieve the effect of optimizing the extraction process and improving the extraction efficiency and purity.
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Embodiment 1
[0064] Example 1 Extraction of 30-50K middle molecular weight thymosin by using the waste in the production process of thymosin
[0065] 1. Raw material processing: Weigh 620kg of thymus tissue, remove fat, ducts and fascia, wash with drinking water for 3 times, and then wash with purified water for 3 times. Control moisture. Pour the cleaned thymus into the colloid mill, add 310L of purified water, start the colloid mill, homogenate for 3 minutes, and import the homogenate into a clean sandwich pot.
[0066] 2. Denaturation: Continue to heat up the homogenate solution to 90°C and keep it for 10 minutes to obtain a denatured solution. After cooling the denatured solution, put it into a stainless steel container for storage.
[0067] 3. Freezing and thawing: Repeated freezing and thawing of the denatured solution 3 times: the freezing temperature is below -20°C, and the melting temperature is 20°C.
[0068] 4. Centrifugation: Thaw the frozen-thawed solution, centrifuge at 400...
Embodiment 2
[0074] Example 2 Utilizes the waste material in the production process of thymosin to extract DNA
[0075] 1, the precipitation C1, C2 among the embodiment 1 and L1, L2 are mixed, and 620kg raw material obtains waste material about 100Kg altogether.
[0076] 2. Add 200L of 2mol / L NaCl solution and 5L of 20% SDS solution, and mix well.
[0077] 3. Heat in a water bath at 55°C for 1 hour, centrifuge at 4000 rpm for 40 minutes, take the supernatant, and discard the precipitate.
[0078] 4. Add 100L chloroform to the supernatant, vortex mix for 4 hours, and let stand overnight to separate.
[0079] 5. The next day, absorb 82L of the supernatant, and add 144L of -20°C 95% ethanol under stirring to produce a white flocculent precipitate, and place it overnight at 4°C.
[0080] 6. Centrifuge at 4°C and 4000rpm for 40min to collect white viscous precipitate.
[0081] 7. Add 50L of 95% cold ethanol, stir vigorously, fully wash the precipitate, centrifuge, and take the precipitate. ...
Embodiment 3
[0084] The active assay of molecular weight thymosin in embodiment 3
[0085] Endothelial cells, mouse 3T3 fibroblast proliferation assay. Endothelial cells were prepared by ourselves, and mouse 3T3 fibroblasts were purchased from Shanghai Yanjing Biotechnology Co., Ltd.
[0086] 1. Preparation of endothelial cells
[0087] 1. Take the umbilical cord of a healthy puerpera within 3 hours after delivery, rinse the umbilical vein repeatedly with D-Hanks solution until there is no blood, pour 5ml of 0.25% trypsin into the 37℃ water bath for 10 minutes, and collect the digestive juice;
[0088] 2. Flush the umbilical vein with 5ml of 1640 culture medium containing 10% fetal bovine serum, and collect the flushing fluid;
[0089] 3. Combine the above digestive juice and washing liquid into a centrifuge tube, and centrifuge at 1000rpm for 10 minutes;
[0090] 4. Remove the supernatant, add an appropriate amount of 1640 culture medium (containing 10% fetal bovine serum, 2000 μg / ml p...
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