Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-08-24
2004-10-12
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S235100, C435S320100, C435S252330, C435S325000, C435S254110, C435S254210
Reexamination Certificate
active
06803214
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to purified and isolated polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, and the use of such nucleic acids and polypeptides in diagnostic methods, kits, vaccines, or antiviral therapy.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) is an important etiologic agent of hepatocellular carcinoma (HCC). However, the mechanism of carcinogenesis by HCV is poorly understood. Although liver cirrhosis caused by the virus may be of primary importance in triggering the malignant transformation of hepatocytes, recent evidence suggested that some HCV proteins have transforming capacities and thus can be implicated in the pathogenesis of HCC (Ray et al., 1996; Sakamuro et al., 1995).
The HCV genome is a plus-stranded RNA about 10 kb in length that encodes a single polyprotein of 3009-3010 amino acids processed co- or post-translationally by both cellular and viral proteinases to produce at least 10 mature structural and non-structural viral proteins. The structural proteins are located in the amino terminal quarter of the polyprotein, and the non-structural (NS) polypeptides in the remainder (for a review see Houghton, 1996). The genome organization resembles that of flavi- and pestiviruses, and HCV is now considered to be a member of the Flaviviraidae family (Miller and Purcell, 1990; Ohba et al., 1996).
The gene products of HCV are, from the N-terminus to the C-terminus: core (p22), E1 (gp35), E2 (gp 70), NS2 (p21), NS3 (p70), NS4a(p4), NS4b (p27), NS5a (p58), NS5b (p66). Core, E1, and E2 are the structural proteins of the virus processed by the host signal peptidase(s). The core protein and the genomic RNA constitute the internal viral core and E1 and E2 together with lipid membrane constitute the viral envelope (Dubuisson et al., 1994; Grakoui et al., 1993; Hijikata et al., 1993). The NS proteins are processed by the viral protein NS3, which has two functional domains: one (Cpro-1), encompassing the NS2 region and the N-terminal portion of NS3, which cleaves autodatalytically between NS2 and NS3, and the other (Cpro-2), located solely in the N-terminal portion of NS3, cleaves the other sites downstream NS3 (Bartenschalger et al., 1995; Hijikata et al., 1993).
One of the characteristics of HCV is its high degree of genetic heterogeneity in vivo, manifested both in the generation of viral quasi-species and in the continuous emergence of neutralization escape mutants (Shimizu et alk., 1994). This poses an obstacle to the development of a broadly reactive HCV vaccine based on antibody reactivity to the envelope glycoproteins (Chien et al., 1993).
Although alpha interferon has been shown to be useful for delaying the development of HCC in chronically infected HCV patients (Nishiguchi et al., 1995), a highly effective therapeutic agent has not yet been developed to control this important infection and to prevent HCC development. For these reasons, there is a considerable need for the development of a detailed understanding of HCV proteins, which should clarify the mechanisms by which HCV induces hepatocyte transformation. Such an understanding may lead to effective means to treat or control the infection, as well as aid in the diagnosis of HCV infection in humans.
SUMMARY OF THE INVENTION
Accordingly, this invention aids in fulfilling these needs in the art. The invention encompasses a purified nucleic acid molecule comprising the DNA sequence of SEQ ID NO:2 and a purified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:1. The invention also encompasses nucleic acid molecules complementary to these sequences.
The invention also encompasses purified polypeptides encoded by these nucleic acid molecules, including purified polypeptides having a molecular weight of approximately 17.5 kD, as predicted by the sequence, hybrid proteins containing amino acid sequences from core and core+1, and purified polypeptides in non-glycosylated form.
The invention includes double-stranded nucleic acid molecules comprising the DNA sequence of SEQ ID NO:2 and purified nucleic acid molecules encoding the amino acid sequence of SEQ ID NO:1. Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention. These molecules can be used to detect both single-stranded and double-stranded RNA and DNA variants of encoding polypeptides encompassed by the invention. A double-stranded DNA probe allows the detection of nucleic acid molecules equivalent to either strand of the nucleic acid molecule.
Purified nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising the DNA sequence of SEQ ID NO:2 or an purified nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:1 under conditions of moderate stringency in 50% formamide and 6×SSC, at 42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% SDS are encompassed by the invention.
The invention further encompasses purified nucleic acid molecules derived by in vitro mutagenesis from SEQ ID NO:2. In vitro mutagenesis includes numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.
The nucleic acid molecules of the invention, which include DNA and RNA, are referred to herein as “core+1 nucleic acids” or “core+1 DNA”, and the amino acids encoded by these molecules are referred to herein as “core+1 polypeptides.”
The invention also encompasses purified nucleic acid molecules degenerate from SEQ ID NO:2 as a result of the genetic code, purified nucleic acid molecules, which are allelic variants of core+1 nucleic acids or a species homolog of core+1 nucleic acids. The invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors.
Purified polyclonal or monoclonal antibodies that bind to core+1 polypeptides are encompassed by the invention.
The invention further encompasses methods for the production of core+1 polypeptides, including culturing a host cell under conditions promoting expression, and recovering the polypeptide from the culture medium. Especially, the expression of core+1 polypeptides in bacteria, yeast, plant, and animal cells is encompassed by the invention.
This invention also provides labeled core+1 polypeptides. Preferably, the labeled polypeptides are in purified form. It is also preferred that the unlabeled or labeled polypeptide is capable of being immunologically recognized by human body fluid containing antibodies to HCV. The polypeptides can be labeled, for example, with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
Immunological complexes between the core+1 polypeptides of the invention and antibodies recognizing the polypeptides are also provided. The immunological complexes can be labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
Furthermore, this invention provides a method for detecting infection by HCV. The method comprises providing a composition comprising a biological material suspected of being infected with HCV, and assaying for the presence of core+1 polypeptide of HCV. The polypeptides are typically assayed by electrophoresis or by immunoassay with antibodies that are immunologically reactive with core+1 polypeptides of the invention.
This invention also provides an in vitro diagnostic method for the detection of the presence or absence of antibodies, which bind to an antigen comprising the core+1 polypeptides of the invention or mixtures of the polypeptides. The method comprises contacting the antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies i
Georgopoulou Urania
Mavromara Penelope
Varaklioto Agoritsa
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Ketter James
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