Preparation method and application of nanoparticle for showing hepatitis C virus envelope protein E2

An envelope protein, fusion protein technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve problems such as reducing the viral load of secondary infection

Inactive Publication Date: 2018-02-02
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, clinical data show that in addition to the high chronic rate, 20-25% of patients can spontaneously clear the HCV virus through early neutralizing antibodies and

Method used

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  • Preparation method and application of nanoparticle for showing hepatitis C virus envelope protein E2
  • Preparation method and application of nanoparticle for showing hepatitis C virus envelope protein E2
  • Preparation method and application of nanoparticle for showing hepatitis C virus envelope protein E2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Example 1 sE2-Ferritin can be successfully secreted and expressed in Drosophila S2 cells

[0165] The recombinant plasmid pMT / Bip / sE2-Ferritin-V5-HisA for secreting and expressing sE2-Ferritin protein in Drosophila S2 cells is as figure 1 As shown in A-C, the truncated secretory segment (384-661) gene of the envelope protein E2 of the standard HCV type 1b strain Con1 was codon-optimized and then linked to the non-heme ferritin carrier gene of Helicobacter pylori for fusion expression, in order to pass the ferritin The HCV envelope protein is correctly displayed on the surface of the ferritin, thereby forming nanoparticles that can self-assemble and secrete in the cell supernatant, and enhance the antigenicity of the envelope protein. In addition, the subunit vaccine recombinant plasmids pMT / Bip / sE2-V5-HisA and pMT / Bip / Ferritin-V5-HisA were used as controls. The sE2-Ferritin and Ferritin gene fragments are followed by a stop codon, while the sE2 protein carries the puri...

Embodiment 2

[0166] Example 2 sE2-Ferritin nanoparticles can be successfully assembled and purified

[0167] To determine whether monomeric sE2-Ferritin can self-assemble into nanoparticles. The present inventors purified the protein sE2 secreted into the supernatant as a control through a nickel column. The sE2-Ferritin and Ferritin secreted into the supernatant were passed through a 10% sucrose pad, resuspended in PBS, and then subjected to 10%-50% sucrose density gradient centrifugation, and divided into 12 layers for SDS-PAGE, ELISA and other tests. Depend on figure 2 A Coomassie brilliant blue staining showed that sE2-Ferritin and Ferritin were distributed in layers 5, 6, 7 and layers 3, 4, and 5, respectively. figure 2 B-C showed that the sE2-Ferritin distribution layer detected by immunoblotting and enzyme-linked immunosorbent assay with HCVE2-specific detection antibody 1C9 and conformational antibody 1B4 were consistent with the results of Coomassie brilliant blue staining. I...

Embodiment 3

[0168] Example 3 The HCVsE2 envelope protein displayed by nanoparticles is near native conformation

[0169] To test whether the self-assembled nanoparticles displayed sE2 close to the native conformation of HCV envelope protein E2, as image 3 As shown in A-B, the inventors used HCV linear neutralizing antibody AP33 and conformation neutralizing antibody AR3A to bind to sE2-Ferritin nanoparticles, respectively. Both sE2 and sE2 can be combined with both antibodies, and the binding ability of nanoparticles is significantly stronger than that of protein, and the binding is dose-dependent. image 3 C shows that sE2 displayed by nanoparticles can bind to HCV receptor CD81, and the binding is significantly higher than that of sE2 protein in a dose-dependent manner, which indicates that the HCV truncated envelope protein displayed by nanoparticles is close to the native conformation. The binding ability of the conformation neutralizing antibody AR3A to sE2-Ferritin nanoparticles a...

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Abstract

The invention provides a preparation method and application of a nanoparticle for showing a hepatitis C virus envelope protein E2. Specifically, a truncated envelope protein sE2 (384-661) gene of an HCV 1B-type Con1 strain and a helicobacter pylori nonheme-iron protein gene are subjected to fusion expression, and sE2 can be shown on the surface of a nanoparticle in a correct conformation by self-assembling of ferroprotein monomers, so that the nanoparticle can be prepared into an HCV nanoparticle vaccine with good antigenicity and high safety, and the immunogenicity of a fusion protein is remarkably improved.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to the preparation and application of a nanoparticle displaying hepatitis C virus envelope protein E2. Background technique [0002] Hepatitis C virus (HCV) is a serious problem that endangers global public health. There are currently more than 170 million chronic HCV infections worldwide, with 3 to 4 million new cases each year. According to statistics, 80% of HCV-infected patients will develop chronic infection, 25% of them may develop liver cirrhosis, and 20% of them may develop liver cancer. [0003] Hepatitis C virus is a single-stranded positive-sense RNA virus of the genus Hepacivirus of the family Flaviviridae, with a genome of about 9.6 kbp. The HCV genome encodes a total of 10 viral proteins, structural proteins including the nucleoprotein Core, envelope glycoproteins E1 and E2, and non-structural proteins including P7, NS2, NS3, NS4A, NS4B, NS5A and NS5...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/29A61P31/14
CPCA61K39/12C07K14/005C07K2319/00C12N2770/24222C12N2770/24234A61K2039/6068
Inventor 黄忠钟劲王雪松颜雨李大鹏屈攀科
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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