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Assembly mechanism of virus-like particle of hepatitis E virus and preparation method thereof

A hepatitis E virus, virus-like technology, applied in the fields of molecular biology and virology, can solve the problems of HEV cell culture and tissue culture failure and other problems

Active Publication Date: 2012-01-18
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Cell culture and tissue culture of HEV have so far been unsuccessful

Method used

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  • Assembly mechanism of virus-like particle of hepatitis E virus and preparation method thereof
  • Assembly mechanism of virus-like particle of hepatitis E virus and preparation method thereof
  • Assembly mechanism of virus-like particle of hepatitis E virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1: State of p239 derived from HEV capsid protein in urea

[0124] p239 (HEV ORF2 aa368-606, its nucleic acid sequence and amino acid sequence are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2). The p239 protein retains the ability of HEV capsid protein to assemble into virus-like particles, and is used to analyze the assembly mechanism of HEV VLP. The p239 protein is mainly expressed in Escherichia coli in the form of inclusion bodies. After washing and purification, most of the p239 protein in the inclusion body was dissolved in 4M urea solution, and then the p239 protein was purified to a purity of more than 95% in a solution containing 4M urea using anion exchange chromatography and hydrophobic interaction chromatography, for subsequent analysis. In addition, E2 protein derived from HEV capsid protein (HEV ORF2aa394-606, the nucleic acid sequence and amino acid sequence of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively) was expressed ...

Embodiment 2

[0126] Example 2: Discovery of key factors for p239 particle assembly

[0127] When purifying p239 protein in 4M urea solution by HIC, ammonium sulfate is needed to increase the hydrophobicity of the protein. Usually, when performing Phenyl FF Sepharose chromatography, the sample solution contains 1M ammonium sulfate, and finally the p239 protein is eluted with a solution containing 0.1-0.2M ammonium sulfate. After elution, the p239 protein is renatured and assembled into particles, typically by removing the denaturant urea in solution. In the present application, the inventors unexpectedly discovered that by increasing the ammonium sulfate in the urea solution to at least 0.3M and standing for a period of time, the p239 protein can assemble into particles in the presence of urea without removing the urea (i.e. , regardless of the presence or absence of urea). The following are the results of the research on the assembly mechanism and process of p239 protein.

[0128] Ren...

Embodiment 3

[0142] Example 3: Final assembly state of p239 particles

[0143] According to the p239 assembly process described in Example 2, the p239 protein was prepared in 4M urea+0.5M (NH 4 ) 2 SO 4 Incubate at 37°C for 12 hours and then dialyze into PBS solution to obtain the final assembled state of p239 particles. The granularity of p239 particles was studied by transmission electron microscope observation, sedimentation rate analysis, molecular sieve chromatography analysis, dynamic light scattering analysis and other methods.

[0144] TEM observation

[0145] The instrument used is a 200kV transmission electron microscope produced by Japan Electronics Corporation, with a magnification of 25,000 times. The obtained p239 particles were negatively stained with 2% phosphotungstic acid pH 7.0, fixed on a carbon-sprayed copper grid, and observed. Electron microscope results see Figure 7 A, where it can be seen that most of the samples present virus-like particles with a diamete...

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Abstract

The invention illustrates an assembly mechanism of a virus-like particle (VLP) of hepatitis E virus (HEV), and provides a method for assembling the VLP by HEV capsid protein or its variants or fragments. In addition, the invention also provides a method for controlling the assembly of the HEV VLP, which controls the assembly of HEV capsid protein or its variants or fragments to the VLP by controlling the salt concentration and / or temperature.

Description

technical field [0001] The present invention relates to the fields of molecular biology and virology. In particular, the present invention clarifies the assembly mechanism of the virus-like particle (VLP) of hepatitis E virus (Hepatitis E Virus, HEV), and provides a method for HEV capsid protein or its variant Or fragments are assembled into the method of VLP. In addition, the present invention also provides a method for controlling the assembly of HEV VLPs, which controls the assembly of HEV capsid proteins or variants or fragments thereof into VLPs by controlling salt concentration and / or temperature. Background technique [0002] Hepatitis E virus is caused by hepatitis E virus (Hepatitis E Virus, HEV) infection, which is one of the main viral hepatitis, and most of them are self-limited. The symptoms of viral hepatitis E are similar to hepatitis A, but the fatality rate is higher and the symptoms are more severe: the fatality rate of pregnant women can be as high as 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12Q3/00A61K39/12A61P1/16A61P31/14
Inventor 夏宁邵李少伟杨春燕鲜阳凌杜海莲张军
Owner XIAMEN UNIV
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