Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1993-10-15
2000-10-24
Wortman, Donna C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
436518, 530350, 536 2372, G01N 33576
Patent
active
061365270
DESCRIPTION:
BRIEF SUMMARY
It has been known for about 15 years that, aside from hepatitis A and hepatitis B, there are additional hepatitides which, in ignorance of the pathogen, were referred to as non-A-non-B-hepatitis. It has meanwhile been assumed that this group of non-A-non-B-hepatitides is caused by at least two different viruses, which are referred to as hepatitis C or E.
The hepatitis C virus is a single-stranded, encapsulated RNA virus with a diameter of about 50-60 nm. The genome consists of about 10,000 nucleotides and it is possible to differentiate between gene regions, which code for structural proteins such as the envelope and the core protein. Such structural proteins can be called C or core, E or envelope and NS. Moreover, the genome comprises the genes for non-structural components of the virus, such as enzymes, etc. Until now, it was known that the genome comprises different proteins; however, the individual, viral proteins are hardly known (Schweizer Medizinische Wochenschrift (1990), vol. 120, pages 117-124).
A partial nucleotide sequence of the hepatitis C virus was published in the European patent application 88.310922 of the Chiron Corporation. However, it was found out that the nucleotide sequence disclosed there codes only for the so-called non-structural proteins. That part of the nucleotide sequence, which codes for the structural proteins, is not disclosed in this reference.
Further DNA sequences of a non-A-non-B-hepatitis virus antigen are disclosed in European patent application No. 89.309261.9.
The European patent application 90.305421.1 of Chiron Corporation discloses the DNA and amino acid sequence of an HCV virus.
The expression of different HCV virus peptides is known from the state of the art. However, they are always expressed as so-called fusion peptides. The background of the expression as fusion protein is that the hepatitis C virus is adapted to the eukaryotic protein biosynthesis and that therefore it is generally assumed that such polypeptides in bacterial systems, such as the E. coli system, can be expressed only as so-called fusion proteins. The part fused on therefore originates from other proteins, such as the .beta.-galactosidase or the superoxide dismutase. However, it is a disadvantage of such fusion proteins that, when these polypeptides are used for detection reactions, cross reactions can occur with the protein portion fused on and lead to wrongly positive results.
It is therefore an object of the present invention to make polypeptides available, which originate from the hepatitis C virus and can be expressed without a fusion portion but, nevertheless, in good yield.
In the present application, fusion proteins are understood to be those polypeptides, which have a significant proportion of a foreign protein, such as .beta.-galactosidase or superoxide dismutase.
By means of the method disclosed here, it was possible to clone and express polypeptides from the structure proteins C (core) and ENV (envelope). It is important that these polypeptides are parts of structure proteins, which occur in the virus particles at the surface or in regions near the surface. By these means, they come into contact with the immune system and thus bring about an immune response. This immune response is essential, on the one hand, for the detection of an infection, for which it is ascertained whether antibodies to the virus are present and, on the other, for immunization as protection against viral infections.
Preferred polypeptides of the present invention are shown in FIGS. 1, 4 and 6.
By means of the disclosed amino acid sequence, shortened polypeptides, which have the same or comparable immunological properties as the polypeptides described here, can be produced without great difficulty. Likewise, the disclosed amino acid sequences can be altered slightly by exchanging a few amino acids, but so that the immunological properties of the polypeptides are retained. Within the scope of the present invention, those polypeptides are therefore preferred, which represent partial sequences of the disclosed
REFERENCES:
patent: 5350671 (1994-09-01), Houghton et al.
Koshy et al., Evaluation of hepatitis C virus protein epitopes for vaccine development, Trends in Biotechnology 14(10):364-369, 1996.
Okamoto et al., Enzyme-Linked Immunosorbent Assay for Antibodies against the Capsid Protein of Hepatitis C Virus with a Synthetic Oligopeptide, Japan. J. Exp. Med. 60(4):223-233, 1990.
Takeuchi et al., Nucleotide sequence of core and envelope genes of the hepatitis C virus genome derived directly from human healthy carriers, Nucl. Acids Res. 18(15):4626, 1990.
Fuchs Klaus
Motz Manfred
Roggendorf Michael
Soutschek Erwin
Roche Diagnostics GmbH
Wortman Donna C.
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