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Protein for preparation of hepatitis E virus-like particles and method

A virus-like particle technology, applied in the fields of molecular biology and virology, can solve problems such as refractory and assembly, and inability to obtain HEV virus-like particles

Active Publication Date: 2014-12-17
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the results show that this protein fragment can be expressed in inclusion bodies, but it is difficult to refold and assemble in vitro, and it is impossible to obtain HEV virus-like particles

Method used

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  • Protein for preparation of hepatitis E virus-like particles and method
  • Protein for preparation of hepatitis E virus-like particles and method
  • Protein for preparation of hepatitis E virus-like particles and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1495

[0255] Construction of embodiment 1.495 protein and mutants thereof

[0256] Using the HEV Xinjiang strain sequence (NC_001434.1) as a template, the ORF2 gene sequence (Invitrogen) was synthesized and connected to the pMD-18T vector to obtain the plasmid pMD-18T-ORF2. Using pMD-18T-ORF2 as a template, 495F as a forward primer (the 5′ end of its sequence introduces a restriction endonuclease Nde I site (CATATG), ATG is the start codon in the E. coli expression system), and 495R is the reverse primer (the 3′ end of its sequence introduces the restriction endonuclease BamHI site), and the PCR reaction is carried out in the PCR thermal cycler (Biometra T3) according to the following conditions:

[0257]

[0258] The amplified product is a DNA fragment of about 1500bp. The PCR product was ligated with a commercially available pMD18-T vector (manufactured by TAKARA Company), and identified by NdeI / BamHI digestion to obtain the positive clone plasmid pMD18-T-495 inserted with the...

Embodiment 2495

[0266] Expression of embodiment 2.495 protein and mutant thereof

[0267] Take out 5 μL of bacterial liquid from the -70°C ultra-low temperature refrigerator, inoculate it into 5 mL of liquid LB medium containing kanamycin, and culture it with shaking at 37°C at 250 rpm until the OD600 reaches about 0.5; then, transfer it to 500 mL of liquid containing kanamycin In the plain LB medium, shake culture at 250 rpm at 37°C for 4-5 hours. When the OD600 reached about 1.5, IPTG was added to a final concentration of 0.4mM, and induced at 15°C, 20°C, or 25°C for 10, 8, or 8 hours at 250 rpm, respectively. The culture was centrifuged at 8000rpm for 5min, the cells were collected, and the cells were ultrasonically disrupted. After the cells were broken, they were centrifuged, and the supernatant and precipitate were taken for detection (using Coomassie brilliant blue staining or Western blot analysis, Western blot analysis using anti-HEVORF2 monoclonal antibody 16D7 (dilution ratio 1:20...

Embodiment 3495

[0273] Purification of embodiment 3.495 protein mutants

[0274] This example takes the 495 protein mutant 1 as an example to illustrate the purification of the 495 protein mutant capable of soluble expression in Escherichia coli.

[0275] After IPTG-induced expression, the bacteria were collected by centrifugation at 8000rpm for 5min, and then the bacteria were resuspended in the lysate according to the ratio of 1g of the bacteria to 10mL of the lysate (20mM Tris buffer pH7.2, 300mM NaCl). Ultrasonic breaker (Sonics VCX750 ultrasonic breaker) was used to treat bacteria (treatment conditions: working time 15min, pulse 2s, pause 4s, output power 55%). The cell lysate was centrifuged at 12,000 rpm at 4°C for 5 minutes (the same below), and the supernatant was discarded and the pellet was discarded (ie, soluble expression).

[0276] Primary purification of saturated ammonium sulfate

[0277] Precipitate the centrifuged supernatant obtained in the previous step with a saturate...

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Abstract

The present invention relates to the field of molecular biology and virology. In particular, the invention relates to a protein capable of being assembled in vitro into hepatitis E virus-like particles, a coding sequence and a preparation method thereof, and the virus-like particles comprising the protein; the protein and the virus like particles can be used for the prevention of or for the treatment of HEV (hepatitis E virus) infection and diseases caused by the HEV, such as hepatitis E and the like. The invention also relates to use of the protein and the virus like particles in the preparation of pharmaceutical compositions and vaccines, the pharmaceutical compositions or the vaccines are used for the prevention or treatment of the HEV infection and diseases caused by the HEV, such as hepatitis E and the like. In addition, the invention also provides a preparation method of the HEV virus like particles.

Description

technical field [0001] The present invention relates to the fields of molecular biology and virology. In particular, the present invention relates to a protein that can be assembled into a hepatitis E virus (Hepatitis E Virus, HEV) virus-like particle (virus-like particle, VLP) in vitro, its coding sequence and preparation method, and the virus containing it The protein and virus-like particles can be used to prevent or treat HEV infection and diseases caused by HEV infection, such as hepatitis E and the like. The present invention also relates to the use of the above-mentioned protein and virus-like particles for preparing a pharmaceutical composition or vaccine for preventing or treating HEV infection and diseases caused by HEV infection such as hepatitis E and the like. In addition, the present invention also provides a method for preparing HEV virus-like particles. Background technique [0002] Hepatitis E virus is caused by hepatitis E virus (HEV) infection, which is ...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/51C12N15/63C12N5/10A61K39/29A61P31/14
CPCA61K39/29C07K14/005C12N2770/28122C12N2770/28123
Inventor 李少伟王楠张晓王凯航郑明华夏宁邵
Owner XIAMEN UNIV
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