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Mongolia astragalus pathogenesis-related protein, crystal thereof, growth method and application

A technology of Astragalus mongolica and related proteins, which is applied in the field of protein crystals, can solve the problems of easy degradation and low stability, and achieve the effect of improving stability, improving stability, and inhibiting the deposition of ECM

Active Publication Date: 2019-07-09
SHANXI UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The second technical problem to be solved by the present invention is to overcome the defects of low stability and easy degradation of the purified A. Crystals of disease-related proteins from Astragalus mongolica with low condition requirements

Method used

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  • Mongolia astragalus pathogenesis-related protein, crystal thereof, growth method and application
  • Mongolia astragalus pathogenesis-related protein, crystal thereof, growth method and application
  • Mongolia astragalus pathogenesis-related protein, crystal thereof, growth method and application

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Embodiment 1

[0070] This embodiment provides a method for the separation and purification of Astragalus mongolica disease process-related protein (AmPR-10), which specifically includes the following steps (see Image 6 ):

[0071] 1. Preparation of crude extract of Astragalus mongolica

[0072] Slice the dried root of Astragalus mongolica, crush it mechanically, and pass through No. 3 sieve of Pharmacopoeia to obtain Astragalus powder. Add extraction buffer (containing 25mM Tris-HCl, 5mM NaCl, pH 8.0) at a solid-liquid ratio of 1:10, and extract at a constant temperature of 55°C for 60min , the Buchner funnel suction filtered to discard the precipitate, and the supernatant was centrifuged at 12,000 rpm for 20 min to obtain the crude extract of Astragalus mongolica.

[0073] 2. Anion exchange chromatography

[0074] (1) Apply the crude extract of Astragalus mongolica to Q Sepharose Fast Flow XK16 / 20 anion exchange chromatography column, Q Sepharose Fast Flow XK16 / 20 anion exchange chromat...

Embodiment 2

[0086] This embodiment provides a method for determining the amino acid sequence of Astragalus mongolica disease process-related protein (AmPR-10), which specifically includes the following steps:

[0087] 1. Protein Spectrum Analysis

[0088] AmPR-10 was identified by Q Exactive Mass Spectrometer from Thermo Fisher. SDS-PAGE electrophoresis separation to obtain the target band, in-gel enzymatic digestion and mass spectrometry detection, the liquid phase conditions are: after 5 minutes of sample injection, first pre-column desalting and then gradient elution through C18 capillary column for peptide separation, mobile phase A contains An aqueous solution of 0.1% formic acid, the mobile phase B liquid is acetonitrile containing 0.1% formic acid, the mobile phase C liquid is an aqueous solution containing 0.1% formic acid, and the flow rate is 200nl / min. The gradient elution method is as follows: the concentration of acetonitrile increases from 10% to 85% in 5-60min, the concent...

Embodiment 3

[0095] This example provides a method for growing crystals of Astragalus mongolica process-related protein (AmPR-10), using Hampton Research protein crystallization screening kits (Index, PEG / ION, SaltRx, Crystal Screen Kit I, II). The crystallization conditions of AmPR-10 protein were initially screened by the hanging drop vapor diffusion method, and initial crystals were obtained under different crystallization conditions. By optimal adjustment, the following growth conditions are preferred:

[0096] 1. Take the protein AmPR-10 solution purified in Example 1, and concentrate the AmPR-10 solution to obtain an AmPR-10 solution with a concentration of (1 mg / mL).

[0097] 2. Growth of tiny protein crystals

[0098] Take 0.3 μL of the concentrated AmPR-10 solution in step 1, mix it with 0.3 μL of the mother solution (100 mM Tris, pH7.5, 20% PEG3350 (w / v)) and drop it on the crystal growth coverslip, and manually invert it On the crystal growth hole, place it at 18°C ​​and grow ...

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Abstract

The invention discloses a Mongolia astragalus pathogenesis-related protein, a crystal thereof, a growth method and an application. The Mongolia astragalus pathogenesis-related protein has any amino acid sequence of (a) to (c), (a) is an amino acid sequence as shown in SEQ ID NO. 1, (b) is an amino acid sequence with at least 90% of homology of the amino acid sequence as shown in SEQ ID NO. 1, or is a homologous compound, derivative and fragment or mutant without losing biological activity of the amino acid sequence as shown in (a) or (b). According to the Mongolia astragalus pathogenesis-related protein, the amino acid sequence of AmPR-10 is firstly disclosed, identification and functional study of the AmPR-10 are facilitated, the application of the AmPR-10 in preparation of a medicine fortreating idiopathic pulmonary fibrosis is further discovered, treatment effects of the AmPR-10 are obvious, the AmPR-10 serves as an extract of natural Chinese herbal medicines, so that the AmPR-10 is wide in source, low in toxicity and suitable for clinical application.

Description

technical field [0001] The invention relates to the technical field of protein crystals, in particular to a process-related protein of Astragalus mongolica and its crystal, growth method and application. Background technique [0002] Chinese herbal medicine is mainly used according to the active ingredients contained in it. In order to make full use of resources and explore new active ingredients, protein ingredients have gradually attracted attention. In recent years, many proteins with biological activity have been found in various Chinese herbal medicines. Separation and purification of active protein components in Chinese herbal medicines, in order to find some new protein sources with biological activity, has become an important direction for many researchers at home and abroad. [0003] Astragalus (Leguminasae), Papilionaceae (P, ilionoideae), Astragalus (AstragalusLinn), Astragalus membranaceus (Fisch.) Bge. and Mongolian Astragalus (Astragalusmembranaceus (Fisch) Bg...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K1/18C07K1/16C12N15/29A61K38/16A61P11/00
CPCC07K14/415A61K38/00
Inventor 任晋宏薛慧清王宏飞王永辉栾智华李敏刘晔冯前进李安平赵建斌陈薛静
Owner SHANXI UNIV OF CHINESE MEDICINE
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