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Anti-tumor bombyxmori Linnaeus cocoon-lysing enzyme inhibitor as well as purification method and application thereof

A cocoon-dissolving enzyme and inhibitor technology, which is applied in the field of biomedicine, can solve the problems of complex components of silkworm decoction, unclear effective substances of anti-liver cancer and their content

Active Publication Date: 2021-08-06
SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Anti-tumor bombyxmori Linnaeus cocoon-lysing enzyme inhibitor as well as purification method and application thereof
  • Anti-tumor bombyxmori Linnaeus cocoon-lysing enzyme inhibitor as well as purification method and application thereof
  • Anti-tumor bombyxmori Linnaeus cocoon-lysing enzyme inhibitor as well as purification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Extraction, separation and purification of stream soluble enzyme inhibitors:

[0037] (1) Extraction of Sanmented Silk Slitzyme Inhibitor: Weighing the amount of white silkworm in line with the quality standard of China Pharmaceutical (2020), and add the appropriate amount of water and cook after properly crushing, and the boiling liquid is filtered after filtering , The filtrate is freeze and dry, add 10 times volume extraction buffer (50.00 mmol·L) -1 , PH 8.0, Tris-HCl buffer), homogenate extraction, centrifugation (10, 464 × g, 10 min), collected supernatant, precipitate repeatedly 2 times, combined with 3 supernatants, freeze dry, and white silkworm Crude protein freeze powder;

[0038] (2) Ammonium sulphate sulfate induced by zombine solurase inhibitor separation: Take a proper amount of Ba Rajluozoi dry powder dissolved in an appropriate amount of extraction buffer solution, centrifugation (10, 464 × g, 10min), collect the supernatant Liquid, slowly added sulfate adj...

Embodiment 2

[0048] Sequence analysis of stream soluble enzyme inhibitor

[0049] Sniworm soluble enzyme inhibitor samples by PAGE electrophoresis, scratch the main zone, and the method of determining the molecular weight of the stream soluble enzyme inhibitor by flight time mass spectrometry (the flight time of the stream soluble enzyme inhibitor is like Figure 4 As shown, it is measured to measure its molecular weight of 13973.63 Da. Application ABI 491A amino acid sequencer was measured by EDMAN degradation by EDMAN degrading enzyme inhibitor sequence SEQ NO: 1 (EDMAN degradation method of n-end 10 amino acids of the zombus solura enzyme inhibitor) Figure 5 Demolved; according to the molecular weight size of the zombus solurase inhibitor, the top 10 amino acid sequences, and the application of the BLASTP program on the search results of the NR database and the silkworm genomic database, determining the zombic silkworm solura inhibitor is a silkworm genome. The truncated protein of the prote...

Embodiment 3

[0053] Dynamics Analysis of Inhibitory Activities in Sanxin Soluid Enzyme Inhibitors

[0054] Formulated 2.00 nmol · ml -1 (28.00μg · ml -1 ) And 3.00 nmol · ml -1 (42.00μg · ml -1 The zream silkworm soluble enzyme inhibitor solution, each takes 1.00 mL and 2.00 mL 60.00 μg · ml -1 Soluilt enzyme solution (equivalent to 2.50 nmol · ml -1 ) Mix, add 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 ), The reaction is terminated with 0.50 mL of 33% acetic acid solution after 5 min, and Tensile A 410 Value, Application Origin 2018 64bit software inhibitor dynamics analysis sub-procedures, directly in MICHAELISMENTEN model, calculate K m , V max , Ki value; zombie solura enzyme inhibitor residual vitality graph Figure 7 As shown, the results showed that in the reaction system containing 5 NM dissolved enzymes, the zombies inhibitors inhibitors inhibit the active linearity in the range of 0.00-5.00 nm, and the two molar suppre...

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Abstract

The invention discloses an anti-tumor bombyxmori Linnaeus cocoon-lysing enzyme inhibitor as well as a purification method and application thereof. The first 10 amino acid sequences of the N terminal of the anti-tumor bombyxmori Linnaeus cocoon-lysing enzyme inhibitor are shown as SEQ NO: 1, the complete amino acid sequence is shown as SEQ NO: 2, and the DNA sequence is shown as SEQ NO: 3. The method comprises the following steps: dissolving bombyxmori Linnaeus water decoction freeze-dried powder in a buffer solution, obtaining an ammonium sulfate precipitate by virtue of an ammonium sulfate step-by-step salting-out method, performing separating by virtue of affinity chromatography which takes the bombyxmori Linnaeus cocoon-dissolving enzyme as a ligand to obtain a preliminarily purified bombyxmori Linnaeus cocoon-dissolving enzyme inhibitor, and performing separating by virtue of gel filtration chromatography to obtain the bombyxmori Linnaeus cocoon-dissolving enzyme inhibitor with higher purity. The high-purity bombyxmori Linnaeus cocoon-dissolving enzyme inhibitor is obtained through rapid protein liquid chromatography separation. The bombyxmori Linnaeus cocoon-lysing enzyme inhibitor is separated and purified from the bombyxmori Linnaeus water decoction for the first time, the defects that the bombyxmori Linnaeus water decoction is complex in component, unclear in anti-hepatoma drug effect substance and content and incapable of adapting to modern clinical injection or local puncture administration are overcome, and the bombyxmori Linnaeus cocoon-lysing enzyme inhibitor has remarkable hepatoma cell proliferation inhibiting activity.

Description

Technical field [0001] The present invention belongs to the field of biomedical technology, and more particularly to an active protein, in particular, to an anti-tumor spmon swollen enzyme inhibitor and a purification method thereof. Background technique [0002] Primary liver cancer is highly malignant, prognosis, high incidence and mortality, has been rising to my country's second cancer killer, and new drugs that have effectively treated liver cancer have always been an urgent solution in the field of biomedical technology. Chinese medicine silkworm is a Bombyxmori Linnaeus 4 to 5 years of larvae infected with Ba clue (Bals.), The lethal dry body is included in the Chinese Pharmacopoeia, with a spasm, hurricane Accessibility, phlegm, etc. Snillets are used alone or with other traditional Chinese medicine to treat multiple tumor disease. Sniwillets in China are used as a private drug for treatment cancer. There are some literature reports on anti-tumor effects on silkworms. The...

Claims

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Application Information

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Patent Type & Authority Applications(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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