A kind of anti-tumor Bombyx mori cocoon-lysing enzyme inhibitor and its purification method and application

A cocoon-dissolving enzyme and inhibitor technology, applied in the field of biomedicine, can solve the problems of anti-hepatic cancer active substances and their content are not clear, and the composition of silkworm decoction is complicated

Active Publication Date: 2022-05-06
SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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Problems solved by technology

[0005] Aiming at the problems in the prior art that the water decoction of Bombyx mori has complex components, and the effective substances for anti-liver cancer and their contents are not clear, the present invention provides an active protein, in particular, it relates to an anti-tumor Bombyx mori cocoon-lytic enzyme Inhibitor and its purification method and application, the extracted and purified anti-tumor Bombyx mori cocoon-lysing enzyme inhibitor has the activity of inhibiting the proliferation of SMCC-7721 human liver cancer cells in vivo and in vitro, and solves the technical problems in the discovery of anti-liver cancer drug candidates in the prior art

Method used

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  • A kind of anti-tumor Bombyx mori cocoon-lysing enzyme inhibitor and its purification method and application
  • A kind of anti-tumor Bombyx mori cocoon-lysing enzyme inhibitor and its purification method and application
  • A kind of anti-tumor Bombyx mori cocoon-lysing enzyme inhibitor and its purification method and application

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Experimental program
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Embodiment 1

[0036] Extraction, isolation and purification of Bombyx mori cocoon-lytic enzyme inhibitor:

[0037] (1) Extraction of Bombyx mori cocoon-dissolving enzyme inhibitors: Weigh an appropriate amount of Bombyx larvae that meet the quality standards of Chinese Pharmacopoeia Part I (2020 Edition), crush them appropriately, add appropriate amount of water to decoct, and filter the decoction to remove slag , the filtrate was freeze-dried, and 10 times the volume of extraction buffer (50.00 mmol·L -1 , pH 8.0, Tris-HCl buffer), homogenate extraction, centrifugation (10,464×g, 10min), collect the supernatant, repeat the extraction of the precipitate twice, combine the supernatant three times, and freeze-dry to obtain the silkworm Crude protein freeze-dried powder;

[0038] (2) Separation of ammonium sulfate step-by-step salting-out method of the cocoon-dissolving enzyme inhibitor of Bombyx larvae: Take appropriate amount of the above-mentioned freeze-dried powder of Bombyx larvae and d...

Embodiment 2

[0048] Sequence Analysis of Cocoon Lysing Enzyme Inhibitor from Bombyx mori

[0049] Bombyx mori cocoon-lysing enzyme inhibitor sample was transferred to membrane after PAGE electrophoresis, and the main band was cut off; the molecular weight of the deadly silkworm cocoon-lysing enzyme inhibitor was determined by time-of-flight mass spectrometry (the time-of-flight simple diagram of the silkworm cocoon-lysing enzyme inhibitor is shown in Figure 4 Shown), the measured molecular weight is 13973.63Da. ABI 491A amino acid sequencer was used to determine the N-terminal 10 amino acid sequence of Bombyx mori cocoon-lysing enzyme inhibitor SEQ NO: 1 (the Edman degradation method sequencing map of the N-terminal 10 amino acids of Bombyx mori cocoon-lysing enzyme inhibitor is as follows Figure 5 shown); according to the molecular weight of the botany cocoon-lysing enzyme inhibitor, the first 10 amino acid sequences of the N-terminal, and the search results of the nr database and the s...

Embodiment 3

[0053] Kinetic Analysis of Inhibitory Activity of Bombyx mori Cocoon Lysing Enzyme Inhibitor

[0054] Prepare 2.00 nmol·mL -1 (28.00μg·mL -1 ) and 3.00 nmol·mL -1 (42.00μg·mL -1 ), take 1.00 mL and 2.00 mL 60.00μg·mL respectively -1 Cocoon-dissolving enzyme solution (equivalent to 2.50nmol·mL -1 ) were mixed, and 2.00, 4.00, 6.00, 8.00, 10.00, 12.00, 14.00, 16.00 and 18.00 μL of substrate BApNA solution (10.00 mmol L -1 ), after incubation for 5 min, stop the reaction with 0.50 mL 33 % acetic acid solution, measure A 410 value, using the inhibitor kinetics analysis subroutine in Origin 2018 64bit software, directly plotting with the MichaelisMenten model, and calculating K m , V max , Ki value; the residual activity curve of Bombyx mori cocoon-dissolving enzyme inhibitor is as follows Figure 7 As shown, the results show that: in the reaction system containing 5 nM cocoon-lysing enzyme, the cocoon-lysing enzyme inhibitor of Bombyx mori is linearly related to the cocoon...

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Abstract

The present invention discloses an anti-tumor Bombyx mori cocoon-lytic enzyme inhibitor and its purification method and application. The first 10 amino acid sequences of the N-terminal of the anti-tumor Bombyx mori cocoon-lytic enzyme inhibitor are shown in SEQ NO: 1, and the full sequence of amino acids is shown in Shown in SEQ NO: 2, the DNA sequence is shown in SEQ NO: 3. The lyophilized powder of the silkworm decoction was dissolved in the buffer solution, and the ammonium sulfate precipitate was obtained by the ammonium sulfate step-by-step salting out method, and the initially purified silkworm was obtained by separation by affinity chromatography using the silkworm cocoon-lytic enzyme as a ligand. The cocoon-lysing enzyme inhibitor is separated by gel filtration chromatography to obtain a higher-purity cocoon-lysing enzyme inhibitor of the botany silkworm, and the high-purity cocoon-lysing enzyme inhibitor of the botany silkworm is obtained by fast protein liquid chromatography. The invention separates and purifies the silkworm cocoon-dissolving enzyme inhibitor from the silkworm decoction for the first time, which solves the problem that the composition of the silkworm decoction is complex, the anti-liver cancer medicinal substance and its content are not clear, and it cannot adapt to modern clinical injection or local puncture The defect of drug administration has significant activity of inhibiting the proliferation of liver cancer cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an active protein, in particular, to an anti-tumor botany cocoon-lytic enzyme inhibitor and its purification method and application. Background technique [0002] Primary liver cancer has a high degree of malignancy, poor prognosis, high morbidity and mortality, and has risen to the second cancer killer in my country. The development of new drugs for the effective treatment of liver cancer has always been an urgent problem in the field of biomedical technology. Bombyx mori (Bombyxmori Linnaeus) larvae of Bombyxmori Linnaeus (Bombyxmori Linnaeus) 4th to 5th instar larvae are infected with Beauveria bassiana (Bals.) Vuillant, and the dead body is dead body. It is included in the Chinese Pharmacopoeia. Pain relief, phlegm and stagnation resolving and other effects. Bombyx mori alone or in combination with other traditional Chinese medicines are used to treat various...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/30C07K1/20C07K1/16A61K38/17A61P35/00
CPCC07K14/43586A61P35/00A61K38/00
Inventor 王厚伟窦彦玲田景振徐凌川
Owner SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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