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Crystal structure of enterovirus 71 3C protease and application thereof

A technology of crystal structure and protease, applied in the field of protease, can solve the problem of lack of inhibitor molecules

Inactive Publication Date: 2013-01-02
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the correct structure of the wild-type 3C protease of enterovirus 71 virus is not yet available, and inhibitor molecules specific for enterovirus 71 3C protease are also very scarce

Method used

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  • Crystal structure of enterovirus 71 3C protease and application thereof
  • Crystal structure of enterovirus 71 3C protease and application thereof
  • Crystal structure of enterovirus 71 3C protease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Expression and purification of enterovirus 71 type 3C protease

[0022] The DNA sequence encoding enterovirus 71 type 3C protease was artificially synthesized and ligated into the pET-28a vector with double restriction sites. Transform Escherichia coli BL21(DE3) competent cells with the recombinant plasmids, pick the obtained transformant clones and transfer them to an appropriate amount of LB medium (add kanamycin to a final concentration of 50 μg / mL), and culture at 37°C to 600nm The absorbance was 0.8, adding isopropyl-β-D-thiogalactopyranoside (IPTG) inducer to a final concentration of 0.5mmol / L, and induced at 20°C for 12h. After collecting the bacteria by centrifugation at 4200r / min for 30min, suspend the bacteria with an appropriate amount of buffer (25mmol / L Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid), pH7.5, 150mmol / L sodium chloride). The bacteria were lysed by sonication, and centrifuged at 16000r / min for 40min to remove sediment and...

Embodiment 2

[0023] Example 2 In vitro activity detection of enterovirus 71 type 3C protease

[0024] Use a fluorescence spectrophotometer to test the enzyme activity. The sequence of the fluorescent substrate polypeptide is Dacyl-KRTATVQGPSLDFE-Edans. The total volume of the reaction system is 100μL, and the buffer is 50mmol / LTris-HCl, pH7.0, 100mmol / L sodium chloride , 1mmol / L Tri-β-chloroethyl phosphate (TCEP), the final protein concentration was 5μmol / L, and the substrate concentration was 50μmol / L. Excited with light at 340nm, the fluorescence at 495nm was detected to gradually increase, the results are as follows figure 1 As shown, it is known that the polypeptide substrate is cleaved.

Embodiment 3

[0025] Example 3 Crystallization and structural analysis of enterovirus 71 type 3C protease

[0026] 3.1. Crystallization conditions:

[0027] Crystallization was carried out by the hanging drop method, the volume ratio of protein solution and crystallization reagent in the crystallization droplet was 1:1, the protein concentration in the protein solution was 12mg / mL, and the crystallization conditions were 100mmol Tris-HCl pH8.5, 25% polyethylene glycol 4000, 0.8mol lithium chloride, the crystals are stored at 16°C.

[0028] In order to obtain the complex formed by the 3C protease and the inhibitor molecule, the protease and the compound were mixed in a molar ratio of 1:5 at 4° C. overnight. The conditions for co-crystallization of protease and compound are the same as when no compound is added.

[0029] 3.2. Data collection and structure analysis:

[0030] Data collection uses SSRF synchrotron radiation, and data processing uses HKL2000 ([5] Z.Otwinowski and W.Minor, "Proce...

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Abstract

The invention relates to a protease, particularly to a crystal structure of an enterovirus 71 3C protease and an application thereof. The enterovirus 71 3C protease is a wild protein without mutation, the crystal structure of the enterovirus 71 3C protease is composed of two domains, 14 beta-pleated sheets and 4 alpha-helixes form typical chymotrypsin sheet, and an active center is formed by a catalytic triad composed of His40, Glu71 and Cys147; and simultaneously, the crystal structure of the enterovirus 71 3C protease is provided with the same C2-D2 loop conformation with other 3C proteases of small ribonucleic acid (RNA) viruses. The crystal structure of the enterovirus 71 3C protease can be applied to preparation of small molecule inhibitors which can interact with specificity of the enterovirus 71 3C protease.

Description

technical field [0001] The invention relates to a protease, in particular to the analysis of the crystal structure of the enterovirus 71 type 3C protease and its application in drug design and development. Background technique [0002] Enterovirus 71 belongs to the Picornaviridae family and is one of the most common pathogens of HFMD. In addition, it can also cause a variety of diseases related to the nervous system, such as sporular pharyngitis, aseptic meningitis, encephalitis and polio-like paralytic diseases, which may be accompanied by severe central nervous system complications or neuroinflammatory disease. Pulmonary edema ([1]McMinn PC.An overview of the evolution of enterovirus 71 and its clinical and public health significance[J].FEMS Microbiol Rev,2002,26:91-107;[2]Weng KF,Chen LL,Huang PN, et al. Neural pathogenesis of enterovirus 71 infection [J]. Microbes Infect, 2010, 12:505-510). [0003] Hand, foot and mouth disease has the characteristics of high epidemic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12Q1/37A61K31/4015A61P31/14C12R1/93
Inventor 林天伟黎健陈晨彭宣嘉蔡期湑高震霆柳立婕
Owner XIAMEN UNIV
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