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Process for the separating gynostemma pentaphylla ribosome inactivation protein, product and encoding gene thereof

A separation method, the technology of Gynostemma pentaphyllum, applied in the direction of genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problem that the coding gene of the separation method has not been reported.

Inactive Publication Date: 2004-03-17
FUJIAN AGRI & FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Gynostemma pentaphyllum has special effects such as anti-cancer and anti-aging, and the research of saponins and flavonoids in it has been widely reported, but there is no report about the separation method, product and RIP coding gene of Gynostemma pentaphylla active protein (hereinafter referred to as Gynostemma pentaphyllum RIP)

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0019] Reagent preparation:

[0020] Extraction buffer: 5mM PBS containing 0.14M NaCl, pH7.4

[0021] Affinity buffer: 20mM Tris-HCl, pH7.5

[0022] Ion exchange buffer: 10mM PBS, pH6.5

[0023] Take a sample of 1000g fresh leaves of Gynostemma pentaphyllum, wash, dry and crush, add extraction buffer, place at 4°C for 1d→filter and centrifuge to obtain extract→slowly add NH 4 SO 4 Solid makes solution NH 4 SO 4 The final saturation reached 60%, overnight at 4°C, centrifuged at 10,000rpm for 10min to obtain the supernatant → add NH 4 SO 4 Solid makes solution NH 4 SO 4 The final saturation reached 70%, overnight at 4°C, centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate → dissolved in distilled water, dialyzed against distilled water until there was no SO 4 2- , and then fully equilibrated with the affinity buffer for dialysis, and centrifuged for 10 minutes to obtain the supernatant → passed through an affinity chromatography column (Blue Sepharose 6 Fa...

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Abstract

The present invention relates to the separation process, product and encoding gene of Gynostemma pentaphylla ribosome inactivating protein (RIP) and belongs to the field of biotechnology. A fast separation and purification system is established, which has the affinity chromatography with blue dye pseudoligand Cibacron Blue 3GA as main part, stepped ammonium sulfate precipitation and cation exchange chromatography. The RIP has molecular weight of 27 KDa and the 19 amino acid sequence of DINFSLAGADGQTYNTFIA. The RIP has homology of 10-37 % with the RIP of other plant and 37-73 % with the RIPof cucurbitaceous plant; tobacco mosaic virus resisting effect of 99 % and suppression to stomach cancer cell. The RIP has homology base level to RIP of cucurbitaceous plant 47-61 % and amino acid level 37-49 %, and has wide medicinal and agricultural application.

Description

Technical field [0001] The invention relates to a Gynostemma pentaphyllum active protein and a gene encoding the protein, which belong to the field of biotechnology, and are particularly related to the field of biopesticide and biomedicine Background technique [0002] According to literature reports, ribosome-inactivating proteins (RIP) have medical value such as anti-tumor and anti-virus, and agricultural value such as anti-plant virus and pathogenic fungus. It is a multi-active substance and has become a domestic However, different RIPs have their own unique functions, and the typical representatives are pokeweed antiviral protein (PAP) isolated from Phytollaca american abroad and PAP isolated from Trichosanthes kirilowii in China. ) isolated trichosanthin (TCS). Gynostemma pentaphyllum has special effects such as anti-cancer and anti-aging. There have been a lot of reports on the saponins and flavonoids, but there are no reports on the separation method, product and RIP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22C07K14/415C12N15/29C12P19/34
Inventor 林奇英林毅吴祖建谢联辉
Owner FUJIAN AGRI & FORESTRY UNIV
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