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Polypeptides for suppressing invasion of hepatitis C virus

A technology for hepatitis C virus and hepatitis C, which is applied in the direction of antiviral agents, peptides, peptides, etc., can solve the problem of inaccessibility, and achieve the effect of convenient preparation and clear target

Active Publication Date: 2010-10-06
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although overexpression of CLDN1 can allow HCV to enter the non-sensitive cell line 293T, a series of related questions remain to be answered: Can the envelope protein of HCV interact with CLDN1? Why are some non-sensitive cells (such as HeLa) still unable to enter HCV after overexpressing CLDN1? In addition, little is known about the molecular mechanism by which CLDN1 mediates HCV entry

Method used

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  • Polypeptides for suppressing invasion of hepatitis C virus
  • Polypeptides for suppressing invasion of hepatitis C virus
  • Polypeptides for suppressing invasion of hepatitis C virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the design of antiviral polypeptide

[0043] The design principle of the antiviral polypeptide is as follows.

[0044] The first step of HCV infecting the host is that the virus's E1 and E2 envelope proteins interact with the host cell surface receptors SR-BI, CD81, CLDN1 and OCLN in turn, and the first step of virus infection is completed through endocytosis and membrane fusion. Theoretically, the specific region of the cell surface receptor has the ability to bind the HCV envelope protein, and the corresponding competitive polypeptide can be obtained by screening the polypeptide library.

[0045] In view of the rationality of the above experimental design, a peptide library was designed based on the amino acid sequences of HCV receptors SR-BI, CD81, CLDN1, and OCLN. Each polypeptide was 18 amino acids long, and two adjacent polypeptides had 5 amino acid repeats. Area (overlap).

[0046] The peptide library contains a total of 113 peptides. Using pseud...

Embodiment 2

[0054] Embodiment 2, synthesis of polypeptide and determination of its chemical properties

[0055] 1. Synthesis of polypeptide 1 and reference polypeptide

[0056] 1. Synthesis of Peptide 1

[0057] Polypeptide 1 was chemically synthesized by American Genscript Company. Polypeptide 1 was dissolved in DMSO, prepared into a 10 mM stock solution, and stored at -20°C.

[0058] 2. Synthesis of control polypeptide

[0059] Control peptide (NH 2 -polypeptide-COOH): SWLRDIWDWICEVLSDFK.

[0060] The control polypeptide is also a sequence derived from CLDN1, the sequence number in the original polypeptide library is No. 60, the position is next to No. 58 and No. 59 polypeptide, and at the C-terminus of No. 58 and No. 59 polypeptide; after screening, the invasion of HCV by the control polypeptide Has no effect.

[0061] The control polypeptide was synthesized by chemical method by American Genscript Company. The control polypeptide was dissolved in DMSO, prepared into a 10 mM sto...

Embodiment 3

[0070] Example 3, the ability of polypeptide 1 to inhibit virus invasion

[0071] One, the preparation of the HCVpp pseudovirus of various genotypes

[0072] 1, the preparation of the HCVpp pseudovirus of H77 genotype (1a)

[0073] (1) Preparation of recombinant plasmid

[0074] According to the sequence in the HCVdb database (http: / / www.hcvdb.org), the H77 gene shown in sequence 11 of the sequence listing was synthesized by Genscript Company. The H77 gene shown in Sequence 11 was inserted into the vector plasmid pcDNA3 (Invitrogen) by cutting the recognition site with HindIII and EcoRI to obtain the recombinant plasmid pcDNA3-H77 (the H77 gene was cloned downstream of the CMV promoter).

[0075] (2) Preparation of the HCVpp pseudovirus of H77 genotype

[0076] 293T cells were seeded in a 10cm cell culture dish one day before transfection, and the cell density was 50% at the time of transfection. pNL4.3-Luc-R - E. - Plasmid and pcDNA3-H77 co-transfected 293T cells, in wh...

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Abstract

The invention discloses polypeptides for suppressing the invasion of hepatitis C virus (HCV). The invention provides the following ten polypeptides: the polypeptide 1 is as shown by the sequence I in a sequence table, the polypeptide II is as shown by the sequence 2, the polypeptide III is as shown by the sequence 3, the polypeptide IV is as shown by the sequence 4, the polypeptide V is as shown by the sequence 5, the polypeptide VI is as shown by the sequence 6, the polypeptide VII is as shown by the sequence 7, the polypeptide VIII is as shown by the sequence 8, the polypeptide IX is as shown by the sequence 9, and the polypeptide X is as shown by the sequence 10. The polypeptides have the following advantages that: the polypeptides consist of 16 to 24 amino acids so as to be short, synthesized chemically and prepared conveniently by recombinant expression; the sequences and the acting sites of the polypeptides are different from those of a conventional HCV invasion suppressor, the target is clear and the specificity suppresses the interaction between an HCV virus cyst membrane and a host acceptor CLDN1; drug resistance is not caused easily; and because the sequences of the polypeptides are originated from human protein CLDN1 rather than HCV virus cyst membrane protein, the polypeptides have a suppression effect on various gene-type HCVs.

Description

technical field [0001] The present invention relates to a polypeptide for inhibiting the invasion of hepatitis C virus. Background technique [0002] Hepatitis C is caused by hepatitis C virus (Hepatitis C virus, HCV) infection, a considerable part of patients after acute infection can be reverted to chronic infection (Chronic hepatitis C), liver cirrhosis (Cirrhosis) and hepatocellular carcinoma ( Hepatocellular carcinoma, HCC) (Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furuta S, Akahane Y, Nishioka K, Purcell RH, et a1.Interrelationship of blood transfusion, non-A, non- B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis Cvirus. Hepatology. 1990 Oct; 12(4 Pt 1): 671-5.). The chronicity rate of HCV infection is very high, ranging from 50% to 85%, which is extremely harmful to the health and life of patients, and the related medical costs are increasing year by year, which has become a serious social and public hea...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/00A61K38/10A61K38/16A61P1/16A61P31/14
Inventor 杨威
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI