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.

Inactive Publication Date: 2016-05-04
TIANJIN TAICHUANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this conventional extraction and separation process of thymosin, the last intercepted part with a molecular weight above 10KD is regarded as waste and thrown away, which not only causes a waste of resources, but also pollutes the environment

Method used

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  • Method for extracting thymosin, medium molecular thymus protein and DNA from thymus tissue

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to thymus extractives, in particular to a method for extracting thymosin, medium molecular thymus protein and DNA from thymus tissue. According to the method, homogenating, degenerating and freezing and thawing are conducted on the animal thymus tissue, supernatant is obtained in a centrifugation mode, ultrafiltration is conducted through an ultrafiltration system with different molecular weight cut offs, medium molecular thymus protein with the molecular weight cut off of 30KD-50KD and thymosin with the molecular weight cut off smaller than 10KD are acquired, in addition, sediment obtained after centrifugation and raffinate abandoned in ultrafitration in the previous process are collected, and extraction is conducted to obtain DNA. According to the method, due to the fact that the extracting technology is optimized, the extracting efficiency and purity of thymosin are improved, waste in the thymosin production technology is comprehensively utilized, and medium molecular thymus protein which has the medicinal value and DNA are extracted.

Description

technical field [0001] The invention relates to a thymus extract, in particular to a method for extracting thymosin, middle molecular weight thymosin and DNA from thymus tissue. Background technique [0002] The thymus is an important immune organ of the human body, an organ that can secrete hormones and produce various cytokines. Peptides and proteins produced by the thymus regulate the immune response. Therefore, research on thymus extracts at home and abroad has attracted widespread attention. In the 1960s, White, Goldstein and their colleagues made many efforts in this field. They purified thymosin from crude thymus extract for the first time and confirmed that the death rate of neonatal thymus-deficient mice was reduced after injection of thymosin. Survival rates increased and cellular immune functions such as resistance to skin grafts were restored. In recent years, dozens of factors with thymus hormone-like activity have been isolated from thymus tissue and blood, ...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/34C12N15/10
CPCC07K14/57581C12N15/1003
Inventor 聂玲玲马骉杨蓉
Owner TIANJIN TAICHUANG BIOTECH CO LTD
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