Method for extracting and purifying pokeweed antiviral protein from pokeweed

An anti-virus and pokeweed technology, applied in the field of medicine, can solve the problems of low purity and lack of high purity of pokeweed anti-viral protein, and achieve good clinical application prospects

Pending Publication Date: 2019-07-16
海南森瑞谱生命科学药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of method for extracting and purifying pokeweed antiviral protein from pokeweed for the deficiencies in the prior art, the purity of the pokeweed antiviral protein prepared by the method is high, solves the problem of extracting pokeweed in the prior art. The low purity of pokeweed antiviral protein and the lack of high-purity pokeweed antiviral protein reference and standard products

Method used

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  • Method for extracting and purifying pokeweed antiviral protein from pokeweed
  • Method for extracting and purifying pokeweed antiviral protein from pokeweed
  • Method for extracting and purifying pokeweed antiviral protein from pokeweed

Examples

Experimental program
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Embodiment 1

[0021] (1) Pokeweed leaves and extract are ground and homogenized at a ratio of 1:1 (w / v), filtered, centrifuged to take the supernatant, ammonium sulfate solid was added, 30% and 85% were salted out twice, respectively, and placed at room temperature overnight.

[0022] (2) Centrifuge to get the precipitate, dissolve the precipitate in Tris-HCl buffer (PH7.5), use DEAE-Sepharose anion column to remove impurities after desalting and filter, the buffer is 10mmol / L Tris-HCL buffer (PH7.5) ; Desalting and concentration, and then passing through SP-Sepharose cation column, stage elution: first use phosphate buffer with 25mmol / L NaCl to elute non-specific adsorption, and then use phosphate buffer with 60mmol / L NaCl to elute the target protein (Pokeweed antiviral protein). Collect sample peaks by ultrafiltration and vacuum freeze-drying. HPLC detection: TSKgel SP-5PW cation column (7.5×75mm); mobile phase is 20mM potassium phosphate buffer (pH7.0), 0-300mM KCl.

Embodiment 2

[0024] (1) Pokeweed leaves and extract were ground and homogenized in a ratio of 1:2 (w / v), filtered, centrifuged to take the supernatant, ammonium sulfate solid was added, 35% and 90% were salted out twice, respectively, and placed at room temperature overnight.

[0025] (2) Take the precipitate by centrifugation, dissolve the precipitate in Tris-HCl buffer (PH7.6), use DEAE-Sepharose anion column to remove impurities after desalting and filter, and the buffer is 10mmol / L Tris-HCL buffer (PH7.6) ; Desalting and concentration and then passing through the SP-Sepharose cation column, stage elution: first use the phosphate buffer with a buffer of 40 mmol / L NaCl to elute the non-specific adsorption, and then use the phosphate buffer with a buffer of 70 mmol / L NaCl to elute the target protein (Pokeweed antiviral protein). Collect sample peaks by ultrafiltration and vacuum freeze-drying. HPLC detection: TSKgel SP-5PW cation column (7.5×75mm); mobile phase is 20mM potassium phospha...

Embodiment 3

[0027] (1) Pokeweed leaves and extract were ground and homogenized in a ratio of 1:3 (w / v), filtered, centrifuged to take the supernatant, ammonium sulfate solid was added, 40% and 95% were salted out twice, and left at room temperature overnight.

[0028] (2) Centrifuge to get the precipitate, dissolve the precipitate in Tris-HCl buffer (PH7.5), use DEAE-Sepharose anion column to remove impurities after desalting and filter, the buffer is 10mmol / L Tris-HCL buffer (PH7.5) ; Desalting and concentration, and then passing through SP-Sepharose cation column, stage elution: first use phosphate buffer with 45mmol / L NaCl to elute non-specific adsorption, and then use phosphate buffer with 85mmol / L NaCl to elute the target protein (Pokeweed antiviral protein). Collect sample peaks by ultrafiltration and vacuum freeze-drying. HPLC detection: TSKgel SP-5PW cation column (7.5×75mm); mobile phase is 20mM potassium phosphate buffer (pH7.0), 0-300mM KCl.

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Abstract

The invention belongs to the field of medicines, and relates to a method for extracting and purifying pokeweed antiviral protein from pokeweed. The method comprises the following steps: extracting total protein from pokeweed leaves, performing precipitation with ammonium sulfate salt to obtain pokeweed antiviral crude protein, dissolving the pokeweed antiviral crude protein in a Tris-HCl buffer solution, performing further purification by adopting anion exchange chromatography and cation exchange chromatography, performing elution to obtain pokeweed antiviral protein solution, and performing ultrafiltration concentration and vacuum freeze drying to obtain the pokeweed antiviral protein with the purity of more than or equal to 99%. The method performs salt precipitation crude purification,and performs a process path of further purification by adopting anion exchange chromatography and cation exchange chromatography so as to obtain the pokeweed antiviral protein with the purity of morethan or equal to 99%, so that the problems of low purity of the extracted pokeweed antiviral protein in the prior art and lack of a high-purity pokeweed antiviral protein reference substance and a standard substance are solved, and the pokeweed antiviral protein has good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to a method for extracting a single active ingredient from medicinal plants, more particularly, to a method for extracting and purifying Pokeweed antiviral protein from Pokeweed. The highest purity of viral protein can be greater than or equal to 99.0%. Background technique [0002] Pokeweed is included in the "Chinese Pharmacopoeia" and "Chinese Materia Medica". Pokeweed plants contain pokeweed antiviral proteins (PAPs). PAPs are a class of isozymes with enzymatic functions. Like ricin A, they belong to ribosome inactivating proteins (RIP). The mechanism of action is mainly to inactivate the ribosome by cleaving a single adenine from the ribosomal RNA. PAPs inhibit various viruses including cytomegalovirus (CMV), influenza virus, poliovirus (PV), herpes virus (HSV), human immunodeficiency virus (HIV), and lymphocytic choriomeningitis virus (LCMV). and killing effect. [0003] As an API, t...

Claims

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Application Information

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IPC IPC(8): C12N9/24
CPCC12N9/2497C12Y302/02022
Inventor 张春发黄超秦人胜邓柳红高海波陈吉良王胜
Owner 海南森瑞谱生命科学药业股份有限公司
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