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Hepatitis C virus (HCV) B-cell epitope peptide PUHI37 and application thereof

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

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 (HCV) B-cell epitope peptide PUHI37 and application thereof
  • Hepatitis C virus (HCV) B-cell epitope peptide PUHI37 and application thereof
  • Hepatitis C virus (HCV) B-cell epitope peptide PUHI37 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Obtaining of Hepatitis C Virus B Cell Epitope Peptide PUHI37

[0020] 1.1 Preparation of polypeptide antigen and polyclonal antibody

[0021]Search the reported full-length protein sequence data of Chinese HCV subtype 1b genotype HCV isolates in the NCBI database, compare the collected sequences, and find out the full-length envelope protein (with hepatitis C virus H77 as the reference standard , Accession No.NC_004102, amino acid residue positions 192-747) consensus sequence. Starting from the 192nd amino acid residue, artificially synthesize a polypeptide with a length of 20 amino acids, 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), and each synthetic polypeptide within this sequence range is 15 amino acids in length, and adjacent polype...

Embodiment 2

[0029] Example 2 Preparation and titer detection of corresponding antibody to protective B cell epitope peptide 534-548 (PUHI37)

[0030] 1.1 Preparation of antibody corresponding to protective B cell epitope peptide 534-548 (PUHI37)

[0031] The artificially synthesized hepatitis C virus B cell epitope peptide PUHI37 epitope peptide was coupled to KLH and BSA respectively (references: Darwish IA, Alzoman NZ, Abuhejail RM, El-Samani TE. Chem Cent J2012;6:125. ), where BSA-coupled epitope peptides were used to detect the serum antibody titer of Balb / c mice after immunization, and KLH-coupled epitope peptide antigens and adjuvants were mixed in equal volumes. Balb / c mice (8-12 weeks old) were immunized with multiple subcutaneous injections on the back of the neck and 29 days, a total of 3 times. Complete Freund's adjuvant for the first time, incomplete Freund's adjuvant for the last two times, 500 μl of emulsified antigen for each mouse, and the antigen amount is 50 μg.

[003...

Embodiment 3B

[0035] Example 3 Detection of Huh7.5 cells infected with 1b genotype subtype HCVpp by antibodies corresponding to B cell epitope peptide 534-548 (PUHI37)

[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 culture incubator. The next day, mix the HCVpp-containing supernatant (20 μl / 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 and 1:100, respectively. , 1:200, 1:400, 1:800), the HCVpp supernatant without antibody and negative serum was also used as a control, and polybrene (polybrene) with a final concentration of 4 μg / ml was added, and the total volume was 100 μl / well (If less than 100 μl is supplemented with DMEM complete medium to 100 μl), incuba...

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Abstract

The invention provides a novel hepatitis C virus (HCV) B-cell epitope peptide PUHI37. An overlapping peptide method is utilized to find out the new linear protective B-cell epitope peptide of HCV envelope protein and obtain protective antibodies capable of neutralizing HCV 1b genotype / subtype HCVpp (HCV pseudotyped particle). The amino acid composition of the epitope peptide recognized by the antibodies is TDVLLLNNTRPPQGN, and the site is 534-548 (using HCV H77 as the reference standard, Accession No.NC_004102). The determination of the protective B-cell epitope peptide provides a new solution for research and development of HCV therapeutic antibodies and HCV preventive vaccines.

Description

technical field [0001] The invention belongs to the field of immunology, in particular, relates to hepatitis C virus B cell epitope peptide PUHI37 and its application. Background technique [0002] Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family. The HCV genome includes a single long open reading frame encoding a polyprotein consisting of about 3000 amino acid residues. Under the action of cellular and viral proteases, the polymeric protein is cleaved into three main structural proteins and several nonstructural proteins necessary for viral replication. [0003] The HCV genome is highly variable, and only 60% of the nucleotide sequences between highly variable isolates are homologous. HCV can be divided into 6 genotypes and different subtypes. HCV1b and 2a genotypes are more common in my country, and 1b is the main type. In some areas, types la, 2b, and 3b have been reported. Type 6 is mainly found in Hong Kong an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08A61K39/42A61P31/14C07K16/10C07K16/06
Inventor 魏来刘如玉
Owner PEOPLES HOSPITAL PEKING UNIV