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Hepatitis c virus B-cell epitope peptide PUHI34 and application thereof

A hepatitis C virus and epitope peptide technology, applied in the field of immunology, can solve the problems of poor treatment effect, high treatment cost and poor curative effect

Inactive Publication Date: 2014-08-20
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For chronic HCV infection, the most important treatment method is the standardized treatment of interferon combined with ribavirin, but the treatment effect for genotype 1 and 4 is poor, and due to obvious side effects and high treatment costs, a large number of HCV Infected people are forced to forego treatment
Liver transplantation caused by HCV infection has become the most important reason for liver transplantation in Western countries, and it is also one of the important reasons in my country. However, standardized treatment regimens are not effective in blocking reinfection of transplanted livers caused by HCV infection. New prevention and treatment options

Method used

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  • Hepatitis c virus B-cell epitope peptide PUHI34 and application thereof
  • Hepatitis c virus B-cell epitope peptide PUHI34 and application thereof
  • Hepatitis c virus B-cell epitope peptide PUHI34 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Obtainment of hepatitis C virus B cell epitope peptide PUHI34

[0020] 1.1 Preparation of polypeptide antigens and polyclonal antibodies

[0021] Search the reported Chinese 1b gene subtype HCV isolate HCV full-length protein sequence data in the NCBI database, and compare the collected sequences to find the full-length envelope protein (using hepatitis C virus H77 as a reference standard , Accession No. NC-004102, amino acid residue positions 192-747) consensus sequence. Starting from the 192nd amino acid residue, a synthetic polypeptide with a length of 20 amino acids is artificially synthesized, and adjacent polypeptides overlap by 10 amino acids (for example, the first sequence is 192-211, the second sequence is 202-221, and so on) . Sequences 404-453, 514-553 and 604-633 include possible binding sites for CD81 (one of the receptors for HCV invading cells). Each synthetic polypeptide within this sequence is 15 amino acids in length, and adjacent polypeptides ...

Embodiment 2

[0029] Example 2 Preparation and titer detection of the corresponding antibody against protective B cell epitope peptide 519-533 (PUHI34)

[0030] 1.1 Preparation of the corresponding antibody to the protective B cell epitope peptide 519-533 (PUHI34)

[0031] Coupling the synthetic hepatitis C virus B cell epitope peptide PUHI34 polypeptide with KLH and BSA respectively (reference: Darwish IA, Alzoman NZ, Abuhejail RM, El-Samani TE.Chem Cent J2012;6:125.), Among them, the BSA-conjugated peptide is used to detect the serum antibody titers of Balb / c mice after the immunization is completed. The KLH-conjugated peptide antigen and adjuvant are mixed in equal volumes. After the emulsification is completed, the first day, the 15th day and the 29th day will be used. Balb / c mice (8-12 weeks old) were immunized with multiple subcutaneous injections on the back for a total of 3 times. The first complete Freund's adjuvant, the last two times are incomplete Freund's adjuvant, each mouse 500μl...

Embodiment 3B

[0035] Example 3 Detection of B cell epitope peptide 519-533 (PUHI34) corresponding antibody neutralizing 1b gene subtype HCVpp infected Huh7.5 cells

[0036] The obtained polyclonal antibody was subjected to HCVpp neutralization experiment. The method includes pre-seeding Huh7.5 cells in a 96-well plate containing DMEM complete medium at a seeding density of 1×10 4 / hole. Incubate in a 37°C cell incubator. On the second day, mix the HCVpp-containing supernatant (20μl / well) with different polyclonal antibodies and negative control serum at different dilution ratios (the dilution ratios of polyclonal antibody and negative control serum are 1:50, 1:100, respectively , 1:200, 1:400, 1:800), HCVpp supernatant without antibody and negative serum is also used as a control, and polybrene (polybrene) with a final concentration of 4μg / ml is added, and the total volume is 100μl / well (Use DMEM complete medium to make up to 100μl for less than 100μl), incubate at 37°C for 1 hour, add to a ...

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Abstract

The invention provides a novel hepatitis c virus B-cell epitope peptide PUHI34. The novel hepatitis c virus B-cell epitope peptide PUHI34 is characterized in that a new linear protective B-cell epitope peptide of the hepatitis c virus envelope protein is found out in an overlapping peptide way, and the protective antibodies capable of neutralizing the hepatitis c virus 1b gene subtype HCVpp (Hepatitis C Virus pseudoparticles) can be gained; the amino acids of the epitope peptides distinguished by the antibodies are formed as TDRFGVPTYNWGENE, and are positioned at 519-533 (refer to hepatitis c virus H77, Accession No.NC-004102). The protective B cell epitope peptide is determined, and thus a novel solution is provided for the study on an HCV therapeutic antibody as well as the study on an HCV prophylactic vaccine.

Description

Technical field [0001] The invention belongs to the field of immunology, and specifically relates to the hepatitis C virus B cell epitope peptide PUHI34 and its application. Background technique [0002] Hepatitis C virus (HCV) is a single-stranded positive-stranded RNA virus belonging to the Flaviviridae family. The HCV genome includes a single long open reading frame encoding a polymerized protein consisting of approximately 3000 amino acid residues. Under the action of cell and viral proteases, the polymerized protein is cleaved into three main structural proteins and several non-structural proteins necessary for virus replication. [0003] The HCV genome is highly variable, and only 60% of the nucleotide sequences are homologous between highly variable isolates. HCV can be divided into 6 genotypes and different subtypes at present. HCV 1b and 2a genotypes are more common in my country, among which type 1b is the main one. Types la, 2b, and 3b have been reported in some regio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K16/10C07K16/06A61K38/10A61K39/29A61P31/14
Inventor 魏来刘如玉
Owner PEOPLES HOSPITAL PEKING UNIV