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Jatropha curcas L. ribosome-inactivating protein and separation and purification method and application thereof

A ribosome inactivation, separation and purification technology, applied in the field of plant protein separation and purification, can solve problems such as lack of targeted therapeutic drugs, and achieve obvious effects and obvious effects of in vitro proliferation inhibition.

Active Publication Date: 2017-08-18
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Malignant tumors are a serious threat to human life and health, but there is still a lack of effective targeted treatment drugs

Method used

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  • Jatropha curcas L. ribosome-inactivating protein and separation and purification method and application thereof
  • Jatropha curcas L. ribosome-inactivating protein and separation and purification method and application thereof
  • Jatropha curcas L. ribosome-inactivating protein and separation and purification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] In this example, it is confirmed by experiments that the Jatropha curcas ribosome inactivation protein Curcin C exists in the cotyledon and endosperm of Jatropha curcas seedlings after germination.

[0047] Soak Jatropha curcas seeds in tap water for 2 hours, then plant them on a culture bed made of vermiculite, germinate at a temperature of 25-30°C, 18 hours of light and 6 hours of darkness, and water properly to keep the culture bed moist , when the cotyledons are fully expanded, the seedlings are transplanted into the mixed soil with a volume ratio of vermiculite and nutrient soil of 1:1, and continue to grow under the same environmental conditions as for germination. From soaking the seeds, the cotyledon and endosperm of Jatropha curcas were collected every day until the cotyledon and endosperm fell off from the Jatropha curcas seedlings, wherein, the cotyledons fell off from the Jatropha curcas seedlings about 49 days after germination, and the endosperm fell off on...

Embodiment 2

[0051] In the present embodiment, from the cotyledons and endosperm of germinated Jatropha curcas seedlings, Curcin C is isolated and purified, and the steps are as follows:

[0052] (1) Preparation of crude extract

[0053] ① Germinate Jatropha curcas seeds, collect 40 g of cotyledons and endosperms of Jatropha curcas seedlings that have germinated for 4 to 49 days, grind the collected cotyledons and endosperms with liquid nitrogen respectively to powder, and then add 120 mL of Dissolve the powdered material in buffer A at 4°C, homogenize at 4°C, homogenize for 30 seconds every time, stop for 30 seconds, and homogenize for 5 minutes, rotate the homogenate at 4°C overnight, and rotate at 4°C at 10,000 rpm Centrifuge at a rotational speed for 30 minutes and collect the supernatant (that is, the homogenate crude extract);

[0054]②Add powdered ammonium sulfate to the supernatant under stirring at 4°C until the saturation of ammonium sulfate in the supernatant is 60%, continue s...

Embodiment 3

[0073] In this example, the N-terminal amino acid residue sequence of Curcin C was determined, and compared with the N-terminal amino acid residue sequence of Jatropha curcas ribosome inactivation protein reported in the existing literature.

[0074] The sequence of the N-terminal amino acid residues of Curcin C was obtained by the following method: the Curcin C obtained in Example 2 was electrophoresed on a denatured polyacrylamide gel, then transferred to a PVDF membrane, and Shimadzu PPSQ-33A type full-body The sequence of the N-terminal amino acid residues of Curcin C was determined by an automatic protein peptide sequencer, and the result was: -Ala Gly Ser ThrPro Thr Leu Ala Ile Thr Tyr Asp Ala Thr Thr.

[0075] Compared the sequence of N-terminal amino acid residues of Curcin C protein with the sequence of N-terminal amino acid residues of RIPs on NCBI, it was found that there were three groups of proteins with sequences close to the N-terminal amino acid residues of Curc...

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Abstract

The invention provides jatropha curcas L. ribosome-inactivating protein and a preparation method thereof. The jatropha curcas L. ribosome-inactivating protein is separated and purified from cotyledon and / or endosperm of jatropha curcas L. seedlings which are generated by jatropha curcas L. seeds germinating for 4 to 49 days, relative molecular weight is 31.398kDa, an isoelectric point is 7.12, and a sequence of amino acid residue of an N-terminal of the ribosome-inactivating protein is shown as SEQ ID No: 1 in a sequence table. The invention further provides application of the jatropha curcas L. ribosome-inactivating protein in preparing medicines for treating tumor. The jatropha curcas L. ribosome-inactivating protein has an obvious anti-proliferative effect on human osteosarcoma U20S, human non-small cell lung cancer A549, human non-small cell lung cancer H1975 and human colon cancer HCT116; an in-vitro proliferation inhibition effect of the jatropha curcas L. ribosome-inactivating protein on the human osteosarcoma U20S is superior to that of Curcin and taxol; furthermore, toxicity to human normal kidney cells is lower.

Description

technical field [0001] The invention belongs to the technical field of separation and purification of plant proteins, in particular to Jatropha curcas ribosome inactivation protein and its separation and purification method and application. Background technique [0002] Malignant tumors are a serious threat to human life and health, but there is still a lack of effective targeted treatment drugs. Plants are rich in various biologically active protein molecules, and it is an effective way to find high-efficiency and low-toxic tumor therapeutic drugs from them. Ribosome-inactivating proteins (ribosome-inactivating proteins, RIPs, EC 3.2.2.22) is a cytotoxic protein present in higher plants or fungi, mostly with RNA N-glycosidase or polynucleotide: adenosyl glycosidase activity , by acting on the N-C glycosidic bond of the conserved sarcin / ricin cyclic adenosine on the ribosomal 28SrRNA, the ribosome is inactivated, thereby inhibiting the progress of protein translation. Acco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24A61K38/47A61P35/00
CPCA61K38/00C12N9/2497C12Y302/02022
Inventor 徐莺杨千张杨雪丁蒙蒙陈放
Owner SICHUAN UNIV
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