HCV envelope protein E2 with deleting hypervariable region 1 and use thereof

A technology of hypervariable region and envelope, applied in the field of biomedical engineering

Inactive Publication Date: 2011-10-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HVR1 antibody can efficiently neutralize HCV infection in vitro, but the neutralization effect of HVR1 antibody has strict strain specificity, that is, the antibody against a certain strain of HCV HVR1 can only neutralize the infection of the same strain of HCV, but not the infection of other strains of HCV. No neutralization

Method used

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  • HCV envelope protein E2 with deleting hypervariable region 1 and use thereof
  • HCV envelope protein E2 with deleting hypervariable region 1 and use thereof
  • HCV envelope protein E2 with deleting hypervariable region 1 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Amplification of the 1-6 genotype HCV envelope E2 protein gene containing HVR1 and deleting HVR1:

[0049] HCV envelope protein E1E2 genes of types 1a, 1b, and 2a were donated by Professor C.M.Rice of Rockefeller University in the United States; HCV envelope protein E1E2 genes of types 3a, 4, 5, and 6 were donated by Professor J.K.Ball of Nottingham University in the United Kingdom. See Table 1 for the gene sequence. Design and synthesize primers according to the corresponding sequences of 7 HCV envelope E2 genes of 1-6 genotypes (1a, 1b, 2a, 3a, 4, 5, 6), and amplify E2 protein by polymerase chain reaction (PCR) Extracellular segment gene (corresponding to 364-661 amino acid residues of HCV polyprotein, 364-383 amino acid residues as secretory signal peptide of E2 protein). The sequences of the amplification primers (upstream primer FP1, downstream primer RP) are shown in Table 2. Reagents for PCR amplification are products of Promega Corporation, USA.

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Embodiment 2

[0058] Embodiment 2: Identification of 1-6 genotype HCV envelope E2 protein expression product:

[0059] Human embryonic kidney (HEK) 293T cells were subcultured with DMEM medium containing 10% fetal bovine serum. When the cells were 80% confluent, the cells were digested with 0.25% trypsin, subcultured and inoculated on 35mm cell culture dishes, and cultured overnight. Combine 4 μg of plasmids constructed above with 7 strains of HCV envelope E2 protein expression plasmids pCI-E2t (including HVR1), pCI-E2tΔ (deleted HVR1) and empty vector pCI-neo with 10 μl of transfection reagent lipid Plastid Lipofectamine (product of Invitrogen, USA) was mixed evenly, and transfected into HEK 293T cells, and the operation was performed according to the instruction. After adding the plasmid-liposome mixture to the cell culture dish, place the cell culture dish in a cell culture incubator at 37°C, 5% carbon dioxide, and saturated humidity, and add 1ml of complete DMEM culture solution contain...

Embodiment 3

[0061] Embodiment 3: Mouse gene immunization and antibody detection:

[0062] Use a plasmid extraction kit (product of Qiagen, Germany) to prepare and purify a large number of HCV envelope E2 protein expression plasmids pCI-E2t, pCI-E2tΔ and empty vector pCI-neo of the above 1-6 genotypes, and dissolve them in PBS buffer (pH 7.4), adjust the concentration to 100 μg / ml, immunize Balb / C mice, immunize 10 mice with each kind of plasmid, inject 0.1 ml of the plasmid solution into the tibialis anterior muscle on each side, and the equivalent dose is 20 μg of the plasmid per mouse. A total of three immunizations were performed with an interval of two weeks. One day before each immunization (the time is 0, 2, 4 weeks respectively, the time of first blood collection is set as 0 week) and every two weeks after the last immunization (a total of 7 times, the time is 6, 8, 10, 12, 14, 16, 18 weeks) Blood was collected from the orbit of the mice, and E2t (including HVR1), E2tΔ (deleted HV...

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Abstract

The invention relates to the technical field of biological engineering. At present, there is no effective vaccine for preventing hepatitis C virus (HCV) infection. The amino terminal of HCV envelope protein E2 has 27 amino acid residues with highest variability, known as hypervariable region 1(HVR1). The invention provides an HCV envelope protein E2 without HVR1, also provides an application of the HCV envelope protein E2 without HVR1 on HCV vaccines and HCV infection immunity diagnostic reagents. Animal immunization tests have discovered that HVR1 in 1-6 genotype HCV envelope protein E2 has substantial inhibition effect against immunogenicity of conservative neutralizing epitope in the envelope protein E2, the effectiveness of inducting broad spectrum neutralizing antibody by the E2 protein is significantly enhanced by deleting HVR1, the HCV envelope protein E2 with deleting HVR1 possibly can be used as an effective target antigen of HCV vaccines. The invention has proved that the HCV envelope protein E2 with deleting HVR1 has strong reaction with E2 antibody in serums of HCV infections and can be used as an immunity diagnostic antigen of HCV infections.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a hepatitis C virus (hepatitis C virus, HCV) envelope E2 protein with hypervariable region 1 deleted and its application in hepatitis C vaccine and immunodiagnostic reagents for HCV infection . Background technique [0002] Hepatitis C virus (HCV) belongs to Flaviviridae and is one of the main pathogenic factors of acute and chronic hepatitis transmitted by blood. HCV is a single-stranded positive-sense RNA virus with only one open reading frame, encoding a protein precursor of about 3010 amino acids, including three structural proteins (core protein Core, envelope protein E1, envelope protein E2) and seven non- Structural proteins (p7 protein, NS2, NS3, NS4A, NS4B, NS5A and NS5B) ( figure 1 ) (see literature: Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol. 2000; 242: 55-8...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/51A61K39/29A61P31/14C12Q1/70C12Q1/68G01N33/68
Inventor 赵平戚中田
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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