Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for preparing thymic peptide alpha 1

A technology of thymosin and recombinant bacteria, which is applied in the field of preparing thymosin α1, can solve the problems of high price, high cost, and low content of active ingredients, and achieve the effect of simple operation and low cost

Active Publication Date: 2014-08-20
DALIAN JINXIU BIOLOGICAL ENG +1
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The tissue extraction method is limited by the source of materials, usually the content of active ingredients in the product is low, which affects the therapeutic effect
Tα1 obtained by chemical synthesis has high purity, but the cost is high and expensive, which limits the clinical application of Tα1
Some researchers have tried to prepare Tα1 through genetic engineering technology, but the obtained recombinant protein needs to be treated with protease, and the purification method uses methods such as affinity chromatography or ion exchange, so the production cost is still high

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing thymic peptide alpha 1
  • Method for preparing thymic peptide alpha 1
  • Method for preparing thymic peptide alpha 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, prepare thymosin alpha 1

[0037] 1. Construction of recombinant plasmids

[0038] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0039] 2. Digest the double-stranded DNA molecule obtained in step 1 with restriction endonucleases NdeI and HindIII, and recover the digested product.

[0040] 3. Digest the pET21a(+) vector with restriction enzymes NdeI and HindIII, and recover the vector backbone of about 5443bp.

[0041] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain the recombinant plasmid pET21-Tα1. According to the sequencing results, the structure of the recombinant plasmid pET21-Tα1 is described as follows: between the NdeI and HindIII restriction sites of the pET21a (+) vector, the 7th to 91st nucleotides from the 5' end of the sequence 2 in the sequence listing are inserted DNA molecules shown.

[0042] 2. Preparation of Thymosin α1

[0043] 1. Introduce the rec...

Embodiment 2

[0058] Example 2, Determination of biological activity of thymosin α1 (cell experiment)

[0059] The biological activity of thymosin α1 prepared in Example 1 was determined by the rosette method (thymosin α1 can activate apoptotic thymocytes in fresh pig thymus to form rosettes with sheep cells).

[0060] 1. Collect fresh porcine thymus, and prepare lymphocyte suspension with PBS buffer.

[0061] 2. Incubate the lymphocyte suspension obtained in step 1 at 45°C for 1 hour (shaking once every 5 minutes), wash the cells with PBS buffer and adjust the cell concentration to (3-5)×10 6 pc / ml, divided into test tubes, 0.2ml per tube.

[0062] 3. Preparation of various solutions

[0063] (1) The thymosin α1 solution obtained in Example 1 was gradiently diluted with PBS buffer to obtain dilutions of various concentrations of thymosin α1 (the concentration of thymosin α1 is calculated by the total protein concentration of the solution).

[0064] (2) Dissolve and serially dilute Zidax...

Embodiment 3

[0086] Example 3, Determination of biological activity of thymosin α1 (animal experiment)

[0087] Experimental animals: 15 healthy piglets aged 8 weeks.

[0088] The breed of piglets was "Du × Chang × Da", purchased from Beijing Daxing Breeding Pig and Poultry Factory.

[0089] The experimental animals were divided into three groups, 5 in each group, and were treated as follows:

[0090] Experimental group-1: intramuscular injection of the thymosin α1 solution prepared in Example 1 (adjust the total protein concentration with physiological saline), and inject 1ml (containing 1mg of total protein) per pig;

[0091] Experimental group-2: Intramuscular injection of Zidaxian solution (prepared with normal saline), 1ml per pig (containing 1mg thymosin α1);

[0092] Experimental group-3: intramuscular injection of the control solution A prepared in Example 1 (adjust the total protein concentration with physiological saline), and inject 1ml (containing 1mg of total protein) per pi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for preparing thymic peptide alpha 1. The method provided by the invention sequentially comprises the following steps of: (1) culturing a recombinant expression vector containing a thymic peptide alpha 1 gene and guiding host bacteria to recombinant bacteria, and collecting bacteria and performing ultrasonication; (2) heating to 75 DEG C and standing for 30 minutes and collecting supernate; (3) performing ultrafiltration with a filter membrane with the cut-off molecular weight of 10000 Dalton, collecting the filtrate, namely the thymic peptide alpha 1 liquor; treating at 75 DEG C for 30 minutes to degenerate and centrifugally remove most mycoproteins; and then using the filter membrane with the cut-off molecular weight of 10000 Dalton to further remove impurities so as to obtain the high purity and high activity final product which is equivalent to thymic peptide alpha 1 (zadaxin) chemically synthesized in biological activity. Compared with a conventional synthetic method or chromatographic purification method, the method for preparing the thymic peptide alpha 1 provided by the invention is simple to operate, and the cost is greatly lowered, so that the method is very suitable for industrial production.

Description

technical field [0001] The invention relates to a method for preparing thymosin α1. Background technique [0002] In 1965, Goldestein et al. isolated a biologically active polypeptide molecule from animal thymus and named it Thymosin. In 1972, Goldestein et al. further purified the component with a main molecular weight of 1-15KD, called thymosin component 5 (Thymosin, TF5), which was used in animal research and clinical observation, and found that this component could compensate for hypofunction of the thymus. . [0003] Thymosin α1 (Thymosin α1, Tα1) is one of the most active polypeptides in the thymosin component 5 (TF5) polypeptide mixture. It has the functions of promoting T-lymphocyte differentiation, maturation and enhancing cellular immunity. At present, its peptides have been synthesized abroad It is used clinically to study the treatment of tumors, hepatitis B, hepatitis C and immunodeficiency, and has achieved good results. Tα1 is an acidic polypeptide composed...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K14/575C07K1/34C12R1/19
Inventor 伏显华刘亭见岑峻宇
Owner DALIAN JINXIU BIOLOGICAL ENG