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Application of king cobra toxin protease inhibitor and its derivatives

A technology of protease inhibitors and derivatives, applied in the field of biomedicine

Inactive Publication Date: 2008-05-28
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, most Kunitz-type protease inhibitors act on either trypsin or chymotrypsin, and few Kunitz-type protease inhibitors act on both trypsin and chymotrypsin

Method used

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  • Application of king cobra toxin protease inhibitor and its derivatives
  • Application of king cobra toxin protease inhibitor and its derivatives
  • Application of king cobra toxin protease inhibitor and its derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1: Separation, purification and activity determination of king cobra venom protease inhibitor

[0105] 1. Separation and purification

[0106] During the separation and purification process, the inhibitory activity against trypsin and chymotrypsin was detected to track:

[0107] The first step, molecular sieve Sephadex G-50 gel filtration: King cobra crude venom 0.5g is dissolved in 3ml phosphate buffer (Na 2 HPO 4 -NaH 2 PO 4 , pH5.8, containing 0.1M NaCl), through Sephadex G-50 gel. The G-50 IV peak has dual inhibitory activities against trypsin and chymotrypsin, the arrow peak in Fig. 1 .

[0108] In the second step, the G-50IV peak was further separated and purified by trypsin affinity chromatography. It has dual inhibitory activity to trypsin and chymotrypsin in the hydrochloric acid elution peak, the arrow peak in Figure 2.

[0109] The third step, HPLC-RP-C 18 Purification of Trypsin Affinity Chromatography Eluted Peaks by Hydrophobic Chromatog...

Embodiment 2

[0114] Embodiment 2: Structural determination and molecular cloning of king cobra venom protease inhibitor

[0115] 1. Structure determination:

[0116] Determination of N-terminal partial amino acid sequence

[0117] The purified main peak protein was subjected to Edman degradation method on an automatic protein sequencer (476A type, product of Applied Biosystems, USA), and the sequence of the N-terminal 20 amino acid residues of the main peak protein was determined.

[0118] Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Detection

[0119] The precise molecular weight of the purified main peak protein was determined by MALDI TOF-MS (Bunker).

[0120] 2. Molecular cloning:

[0121] Construction of cDNA Library of King Cobra Venom Gland

[0122] 1) Extraction of total RNA from king cobra venom glands: three days after taking venom from live king cobras, put them into liquid nitrogen for 4 hours and then peel off the venom gland tissues, we...

Embodiment 3

[0127] Embodiment 3: the prokaryotic expression of recombinant king cobra venom protease inhibitor

[0128] Expression of recombinant protein in Escherichia coli

[0129] 1. The cloned king cobra venom trypsin / chymotrypsin bifunctional inhibitor (OH-TCI) was amplified by PCR, and the 5'-terminal primer used corresponds to the N-terminal of the mature polypeptide, and the 3'-primer corresponds to the mature polypeptide C-terminus, containing restriction endonuclease Hind III cleavage site and protected nucleotides. PCR amplification was performed using high-fidelity DNA polymerase Pyrobest, a product of Treasure Bioengineering (Dalian) Co., Ltd. After the product was recovered, it was digested with restriction endonuclease Hind III, and then ligated with the expression vector (pMAL-p2X) cut with restriction endonuclease Xmn I and Hind III for blunt-sticky ends, and transformed into E. coli DH5α in cells. The expression vector pMAL-p2X was purchased from New England Biolabs. ...

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PUM

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Abstract

The invention relates to a king cobra viper venom inhibitor, and relative derivative. Compared with other natural low-molecular-weight prolease inhibitor, the inventive inhibitor has simple structure, high gene expression yield, and special activity. The preparation method of the king cobra viper venom inhibitor can be prepared by separating and purifying crude venom or prepared by gene engineering. The invention further provides the application of the king cobra viper venom inhibitor and relative derivative, which has double inhibition activity of parenzyme and chymotrypsin, in same magnitude order as the inhibition constant on two proleases, and the inhibition activity with sodium channel.

Description

technical field [0001] The invention relates to the application of a king cobra venom protease inhibitor and its derivatives, belonging to the field of biomedicine. Background technique [0002] Protease inhibitors are proteins or small peptides that widely exist in animals, plants and microorganisms and can inhibit the hydrolytic activity of proteases. Protease inhibitors can bind to the active site and / or allosteric site of protease, thereby inhibiting the catalytic activity of the enzyme and / or the binding of the protease to the natural substrate, preventing the zymogen from being converted into an active enzyme, and thus regulating protease activity and plays an important role in substance metabolism. It also plays a key regulatory role in a series of physiological and pathological processes. Protease inhibitors have the prospect of being developed into new drugs that are beneficial to human health. They have shown great promise in the treatment of AIDS, pancreatitis, ...

Claims

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Application Information

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IPC IPC(8): C07K14/81A61K38/57A61P1/18A61P7/04A61P31/18
Inventor 李文辉张云何英英刘树柏
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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