Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-02-09
2001-11-27
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C530S350000, C530S806000, C530S826000, C435S007100, C435S069100, C435S069300, C435S069700, C435S440000, C435S455000, C435S471000, C436S501000, C436S536000, C436S811000, C436S820000, C536S023100, C536S023400, C536S023720
Reexamination Certificate
active
06322965
ABSTRACT:
BACKGROUND OF INVENTION
1. Field of Invention
The present invention relates to an antigen peptide which is capable of detecting a wide range of infection by hepatitis C virus (HCV).
2. Related Art
Non-A, non-B hepatitis is an infectious hepatitis which is believed to be transmitted by means of virus as an agent. Although much remains to be known about the transmission route of non-A, non-B hepatitis, transfusion- or blood products-derived non-A, non-B hepatitis represents a serious medical problem as post-transfusion hepatitis.
In 1989 part of the viral gene associated with non-A, non-B hepatitis was cloned and was designated as hepatitis C virus (HCV) (1989, Choo Q. -L. et al., Science 244: 359-362). At about the same time many HCV genes were isolated by many research groups including the applicant of the present invention, leading to the elucidation of structural features thereof.
The deduced HCV gene has a+strand RNA composed of about 9,300 to 9,500 nucleotides as the genome and encodes one stretch of polypeptide having about 3,000 amino acids. The predicted amino acid sequence thereof has a homology with flavivirus or pestivirus and, thereby HCV virus is believed to be an allied virus thereof. From its similarity to the structures of these viruses, it is believed that the polypeptide encoded by the HCV genome, after being synthesized as a single polypeptide in the cell, is cleaved from the amino-terminal into the structural proteins, core, envelope (E1), and NS1 or E2 (NS1/E2), and the non-structural proteins, NS2, NS3, NS4, and NS5, and then each plays its respective role (1991, Houghton, M. et al., Hepatology 14: 381-391).
Conventional methods used to investigate HCV infection involve the use of these polypeptides encoded by the HCV genome to detect antibodies induced within the patient's body. In particular, since antibodies against core, NS3, and NS4 have been found in many patients with HCV, reagents for detecting antigen-antibody reactions have been prepared using as the antigen part of these polypeptides which were produced by means of genetic engineering, and have played a role in identifying the presence of HCV infection.
However, there is no known antigen which is a single polypeptide comprising a combination of epitope peptides alone and which permits a sensitive detection of a wide range of HCV infection.
SUMMARY OF THE INVENTION
The present invention provides an antigen peptide which permits sensitive detection of a wide range of hepatitis C virus (HCV)infection, a method of producing said antigen, and a method of detecting HCV infection using said antigen peptide.
In order to solve the above problems, the present invention provides a chimera antigen peptide against an HCV antibody comprising a polypeptide wherein at least two or more peptide regions from each of the core region, the NS3 region, and the NS4 region of the HCV polypeptide have been joined.
Thus, the present invention provides an HCV chimera antigen peptide comprising at least two epitope peptide regions of the core region, two epitope peptide regions of the NS3 region, and at least two epitope peptide regions of the NS4 region of the HCV polypeptide. The HCV chimera antigen peptide of the present invention specifically binds to antibodies produced by humans.
More specifically, the present invention provides a chimera antigen peptide comprising three polypeptide regions (C-1, CI, and CII) contained in the core region, two amino acid regions (NS3-1 and NS3-2) contained in the NS3 region, and four amino acid regions (NS4-I1, NS4-I2, NS4-II1, and NS4-II2) contained in the NS4 region of the HCV polypeptide provided that CI, NS4-I1, and NS4-I2 are derived from type I HCV, and CII, NS4-II1 and NS4-II2 are derived from type II HCV. The present invention also provides a chimera antigen peptide which has an amino acid sequence having a homology of 80% or more with the above amino acid sequence.
Preferably the present invention provides a chimera antigen peptide wherein C-1 comprises the amino acid sequence 1-43 (SEQ ID NO: 36) of the HCV polypeptide, CI comprises the amino acid sequence 66-80 (SEQ ID NO: 37) of the type I HCV polypeptide, CII comprises the amino acid sequence 66-80 (SEQ ID NO: 38) of the type II HCV polypeptide, NS3-1 comprises the amino acid sequence 1238-1313 (SEQ ID NO: 39) of the HCV polypeptide, NS3-2 comprises the amino acid sequence 1363-1460 (SEQ ID NO: 40) of the HCV polypeptide, NS4-I1 comprises the amino acid sequence 1712-1750 (SEQ ID NO: 41) of the type I HCV polypeptide, NS4-I2 comprises the amino acid sequence 1678-1705 (SEQ ID NO: 42) of the type I HCV polypeptide, NS4-II1 comprises the amino acid sequence 1716-1750 (SEQ ID NO: 43) of the type II HCV polypeptide, and NS4-II2 comprises the amino acid sequence 1690-1713 (SEQ ID NO: 44) of the type II HCV polypeptide. The present invention also provides a chimera antigen peptide which has an amino acid sequence having a homology of 80% or more with the above amino acid sequence.
More specifically, the present invention provides an HCV chimera antigen peptide which comprises: a single polypeptide comprising the peptide region from position 1238 to position 1311 or from position 1238 to position 1313, the peptide region from position 1363 to position 1460, the peptide region from position 1712 to position 1751, the peptide region from position 66 to position 80, and the peptide region from position 1686 to position 1704 of the HCV polypeptide belonging to the genotype group 1; the peptide region from position 1716 to position 1751, the peptide region from position 66 to position 80, and the peptide region from position 1690 to position 1714 of the HCV polypeptide belonging to the genotype group 2; and, the peptide region from position 1 to position 43 or from position 1 to position 42 of the HCV polypeptide belonging to the genotype group 1 or 2, wherein the above regions may be joined by a linker peptide having no epitope activity; or a single polypeptide which has an amino acid sequence having a homology of 80% or more with the above amino acid sequence.
The present invention also provides DNA encoding the above chimera antigen peptide.
Furthermore, the present invention provides a vector, particularly an expression vector, comprising the above DNA.
The present invention also provides a host which has been transformed by the above expression vector.
Furthermore, the present invention provides a method of producing a chimera antigen peptide comprising culturing the above host, and harvesting said chimera antigen peptide from said culture.
The present invention also provides a method of detecting HCV infection comprising contacting a test sample with the above chimera antigen peptide, and determining whether an antigen-antibody reaction has taken place between them.
In accordance with the technology illustrated by the present invention, the non-specific reactions between antigen and antibody, which always provide problems in the methods employing antigen-antibody reactions, can be reduced, the reactivity of specific reactions between antigens and antibody can be enhanced, and enhanced function of a peptide antigen can be applied to immunological diagnostic methods employing antigen-antibody reactions.
Furthermore, the use of a polypeptide illustrated by the present invention enables detection of antibody to HCV in the serum of patients with HCV infection and thereby is useful for correct identification of the presence of HCV infection.
In accordance with the present invention, the performance, which could only be attained by the production of multiple antigens in the conventional methods, can be attained by the use of one antigen. This has led to a higher efficiency in production such as reduced number of production steps, simplified methods of production, and reduced numbers of tests and simplified testing related to production such as quality control as compared to the conventional methods.
DETAILED DESCRIPTION
Since part of the gene fragment encoding the information required for HCV to propagate was isolated prior
Chiba Yukie
Hasegawa Akira
Kashiwakuma Tomiko
Yagi Shintaro
Yamaguchi Kenjiro
Advanced Life Science Institute, Inc.
Foley & Lardner
Zeman Mary K.
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