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Hepatitis B virus binding proteins and uses thereof

a technology binding proteins, which is applied in the field of hepatitis b virus binding proteins, can solve the problems of high risk of liver cirrhosis and primary liver carcinoma, no antiviral drugs with good and long-term efficacy, and no molecules of these molecules have been convincingly tied

Inactive Publication Date: 2003-10-02
YEDA RES & DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The identified proteins demonstrate specific binding to HBV particles and hepatocytes, providing potential therapeutic agents and cofactors in HBV infection, and can be used to inhibit HBV attachment to hepatic cells.

Problems solved by technology

Infected patients develop acute hepatitis, which is often self-limiting, but may develop into chronic hepatitis with high risk of liver cirrhosis and primary liver carcinoma in roughly 10% of all cases.
Effective vaccines exist, but anti viral drugs with good and long term efficacy are not available.
None of these molecules have been convincingly tied to infectivity, disqualifying them as genuine HBV receptors.
It will be appreciated that so far culture propagation of HBV is impractical.
Alternatively, such hybrid formation may lead to interference with correct splicing.
Unmodified oligonucleotides are typically impractical for use as antisense sequences since they have short in vivo half-lives, during which they are degraded rapidly by nucleases.
Furthermore, they are difficult to prepare in more than milligram quantities.
In addition, such oligonucleotides are poor cell membrane penetraters.
In addition, in order to improve half-life as well as membrane penetration, a large number of variations in polynucleotide backbones have been done, nevertheless with little success.
Such molecules bind to the targeted gene molecules in RNA of tumor cells, thereby inhibiting the translation of the genes and resulting in dysfunctional growth of these cells.
However, due to their low stability RNA oligonucleotides are typically expressed inside the cells using vectors designed for this purpose.

Method used

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  • Hepatitis B virus binding proteins and uses thereof
  • Hepatitis B virus binding proteins and uses thereof
  • Hepatitis B virus binding proteins and uses thereof

Examples

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example 1

Materials and Methods

[0143] Preparation of the affinity columns: For the preparation of the affinity columns first a recombinant preS1 protein was prepared. The HBV preS1 gene was obtained by PCR amplification from a plasmid containing the entire HBV genome (cloned at the laboratory of W. J. Rutter at the UCSF). The following primers were used for amplification: 5'-GGAGATCTTCAAAACCTGGCAAAGGC-3' (SEQ ID NO:10) and 5'-GAATTCCACTGCATGGCCTG-3' (SEQ ID NO:11). The PCR product was cloned into the p-RSET-B vector (Invitrogene). The constructed plasmid was sequenced using the Weizmann Institute service center (see, SEQ ID NO:7). Recombinant His tagged pre-SI protein (see SEQ ID NO:8) was expressed in E. coli B121 cells. Cells were grown overnight at 37.degree. C. in M9ZB medium containing 0.4% glucose. The overnight culture was then diluted 1:50 with fresh M9ZB medium and was further grown at 37.degree. C. When the OD(600 nm) reached 0.7-0.8 the cells were induced with IPTG (1 mM). The solu...

example 2

Production of the Recombinant HBV preS1 Protein

[0154] Based on previous research, the preS1 region of HBsAg is expected to contain the receptor binding region (Neurath et al., 1985; Petit et al., 1991). For HBV receptor purification a recombinant His-tagged preS1 protein (FIGS. 1a and 1b, SEQ ID NOs:7-8) was prepared. The recombinant protein was purified to homogeneity by employing a Ninta-affinity column (Quiagene). Also, a 29 amino-acid long peptide that was reported to be sufficient to interact with hepatocytes was synthesized (SEQ ID NO:9). This synthetic peptide was further purified on a G25 column to obtain a homogenous peptide (FIG. 1c).

example 3

Purification of HBV preS1-Binding Proteins

[0155] The recombinant preS1 protein (SEQ ID NO:7) was covalently cross-linked to beads (Affinity gel 10, BioRad) according to the manufacturer's instructions and was used for affinity chromatography. Concentrated urine (X 1000) was passed through the column, the column was washed and the bound proteins were eluted at low pH (see methods). The eluted fractions were analyzed on SDS-PAGE gel and silver stained. Two major bands appeared after elution from the preS1 column (FIG. 2, lane E2). The estimated molecular masses of the stained proteins were 50 and 43, and therefore they were named UP50 and UP43, respectively.

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Abstract

An isolated nucleic acid, a recombinant protein encoded thereby and uses thereof. The isolated nucleic acid including (a) a polynucleotide at least 60% identical to SEQ ID NOs:1, 3, 5 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3); (b) a polynucleotide encoding a polypeptide being at least 60% homologous with SEQ ID NOs:2, 4, 6 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3); or (c) a polynucleotide hybridizable with SEQ ID NOs:1, 3, 5 or portions thereof at 68° C. in 6xSSC, 1% SDS, 5x Denharts, 10% dextran sulfate, 100 mug / ml salmon sperm DNA, and <32>p labeled probe and wash at 68° C. with 3xSSC and 0.1% SDS.

Description

[0001] This is a divisional of U.S. patent application Ser. No. 09 / 409,096, filed Sep. 30, 1999.FIELD AND BACKGROUND OF THE INVENTION[0002] The present invention relates to a group of genes, and the proteins encoded thereby, which are capable of interfering with Hepatitis B virus (HBV) infection and to methods for identifying, purifying, isolating and characterizing related genes and gene products. The present invention further relates to isolation of soluble forms of the cellular receptor(s) for HBV on hepatocytes from bodily fluids, including, but not limited to, urine, and to purification of these soluble form binding proteins by means including, but not limited to, affinity columns. The present invention further relates to the use of these genes and their translation products to establish experimental models for HBV infection, whether in cell culture or in animals. The present invention further relates to the use of these genes and their translation products for therapeutic purp...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/47
CPCC07K14/47A61K38/00
Inventor SHAUL, YOSEFZEMEL, ROMI
Owner YEDA RES & DEV CO LTD
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