Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not

A technology for thymosin and missing peptides, which is applied in the field of identification of whether purified thymosin α1 contains missing peptides, quality detection of thymosin α1, can solve the problem that there is no public method for measuring thymosin α1 missing peptides, thymosin α1 is not disclosed, and it is not suitable for clinical practice of thymosin α1 Status and other issues

Active Publication Date: 2014-09-24
永春县产品质量检验所福建省香产品质量检验中心国家燃香类产品质量监督检验中心福建
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no disclosure of the missing peptide status in artificial solid-phase synthesized thymosin α1 at home and abroad, and there is no public method for determining the missing peptide in artificial solid-phase synthesized thymosin α1
This state does not accommodate the clinical status of thymosin α1

Method used

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  • Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not
  • Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not
  • Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Solid-phase synthesis of thymosin α1

[0025] 1. Preparation of Fmoc-Asn(Trt)-Wang Resin

[0026] Weigh 10g (3.1mmo1) of Wang Resin and place it in a solid-phase reaction synthesis column. Add 60ml of DCM to swell the resin for 10 minutes, and then remove the DCM. Wash the resin with DMF three times, 50ml each time, blow air for two minutes, and remove the solvent. Weigh 5.5g (9.3mmo1) Fmoc-Asn(Trt) and 1.38g (10.23mmol) HOBt in a ground flask, add 50ml DMF to dissolve. Add 2.15ml (13.95mmol) DIC to activate 5min under cooling in an ice water bath. The activated amino acid was added to the resin washed with DMF, and then 0.23 g (1.86 mmol) of DMAP was added, and the mixture was stirred with nitrogen gas, and reacted at room temperature for 2-4 hours. After the reaction, the resin was washed three times with DMF, 50 ml each time, blowing air for two minutes, and draining the solvent. Take a small amount of resin, shrink it three times with methanol, dry it in v...

Embodiment 2

[0049] Example 2 Purification of thymosin α1 by solid phase synthesis

[0050] 1. One-time purification of thymosin α1 by solid phase synthesis

[0051] Waters 2487 detector, Waters 600 pump, Dima C 18 Preparation column (10μm, 250×21.2mm), ultraviolet detector, wavelength 215nm, TFA / acetonitrile / water gradient elution, the elution gradient is listed in Table 1.

[0052] Table 1 Elution gradient of one-time purification of crude thymosin α1

[0053]

[0054] Dissolve 6.27 g of solid-phase synthetic thymosin α1 crude product in 250 ml of water, first adjust pH 8 with ammonia, then adjust pH 5 with acetic acid, filter, and apply the filtrate to the column. According to the gradient elution in Table 1, the fractions were collected, freeze-dried, and the obtained solid was subjected to secondary purification.

[0055] 2. Secondary purification of thymosin α1 by solid phase synthesis

[0056] Waters 2487 detector, Waters 600 pump, Dima C 18 Preparative column (10μm, 250×21.2mm), UV detector,...

Embodiment 3

[0060] Example 3 Identification of missing peptide map after secondary purification of thymosin α1 by solid phase synthesis

[0061] Take a small amount of solid-phase synthetic thymosin α1 after secondary purification to obtain pure product in LC (Agilent 1200 automatic sampling, Waters Xterra RP18 analytical column, 5μm, 3.0×150mm) / MS (Bruker Solatix FT-ICR-MS, ion current detection Analyze on the combined instrument. The gradient was eluted with 0.1% formic acid / acetonitrile, and the elution gradient is listed in Table 3. Ion current spectrum see figure 1 . figure 1 It consists of 3 peaks, which appear at 20.78min, 22.30min and 27.19min respectively. Their peak areas are 0.36%, 99.16% and 0.47%, respectively.

[0062] Table 3 The elution gradient of pure thymosin α1

[0063]

[0064] The corresponding mass spectrum of the peak appearing at 22.30 min is shown in figure 2 , The mass of the ion is 1036.88553×3, which is thymosin α1

[0065] Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-G...

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Abstract

The invention discloses a method for identifying whether purified thymosin alpha 1 contains deletion peptide or not. The method includes the following steps that (1) a first purification and a second purification are conducted to solid-phase synthetic thymosin alpha 1; (2) gradient elution is conducted to the synthetic thymosin alpha 1 purified products which are purified for two times to obtain ion current spectrums by utilizing an LC / MS mass spectrometer; if the obtained ion current spectrums present peaks at 20.78 min or present peaks at 27.19 min, wherein the areas of the peaks which are presented at 20.78 min are 0.36 % and the areas of the peaks which are presented at 27.19 min respectively are 0.47%, thus the purified thymosin alpha 1 contains the deletion peptide. The method can be accurately and quickly used for identifying whether the thymosin alpha 1contains the deletion peptide or not. Quantity of the thymosin alpha 1 can be effectively guaranteed and controlled. The method for identifying whether purified thymosin alpha 1 contains the deletion peptide or not has important application values in preparing high-purity solid-phase synthetic thymosin alpha 1.

Description

Technical field [0001] The invention relates to a method for quality detection of thymosin α1, in particular to a method for identifying whether purified thymosin α1 contains missing peptides, and belongs to the field of solid-phase synthesis and detection of thymosin α1. Background technique [0002] Thymosin α1 is an active polypeptide isolated from Thymosin component 5 (TF-5), which consists of 28 amino acid residues and has a molecular weight of 3108.37. In TF-5, the content of thymosin α1 is 0.6%, which is an important active component of human thymus hormone. For example, thymosin α1 can regulate the development, differentiation and maturation of T lymphocytes. In addition, thymosin α1 can repair damaged T lymphocytes. Although the thymosin α1 isolated from TF-5 has no obvious toxic and side effects, the commercially available thymosin α1 synthesized by artificial solid phase can cause adverse reactions due to impurity. [0003] At present, the operating procedures of arti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02C07K14/575C07K1/04
Inventor 朱正兵彭涛王玲张金花
Owner 永春县产品质量检验所福建省香产品质量检验中心国家燃香类产品质量监督检验中心福建
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