Check patentability & draft patents in minutes with Patsnap Eureka AI!

Hcv replicon shuttle vectors

A technology of shuttle vector and replicon, applied in the direction of introducing foreign genetic material, virus/phage, viral peptide, etc. using vector, can solve the problem that the replicon cannot establish HCV replication and so on.

Inactive Publication Date: 2007-06-27
F HOFFMANN LA ROCHE & CO AG
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inability of replicons derived from HCV-H to establish efficient HCV replication suggests that the replicons constructed earlier by Lohmann (1999) above depended on the specific genotype 1b consensus cDNA clones used in those experiments

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Hcv replicon shuttle vectors
  • Hcv replicon shuttle vectors
  • Hcv replicon shuttle vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Plasmid construction

[0054] The transient HCV genotype 1b Con1 replicon vector rep PI-luc / ET is shown in Figure 1. This vector was obtained from R. Bartenschlager and contains the HCV polynucleotide sequence published in GenBank Accession No. AJ238799 from 5'-UTR, NS3 to NS5B proteins to 3'-UTR. It also includes the poliovirus internal ribosome entry site (IRES) that controls translation of the firefly luciferase gene. Downstream of the firefly luciferase gene, an IRES derived from encephalomyocarditis virus (EMCV) controls the translation of HCV nonstructural genes (NS3, NS4A, NS4B, NS5A, and NS5B).

[0055] The transient replicon repPI-luc / ET vector was modified by replacing the pBR322 backbone with the pUC18 backbone, resulting in the replicon pPI-luc / ET / SC (SEQ ID NO: 1). In assay assays, pPI-luc / ET / SC replicated at similar levels to repPI-luc / ET (see Table 1).

[0056] The transient HCV genotype 1a H77 replicon vector pSS-1 was obtained by ligating the SpeI-Bs...

Embodiment 2

[0063] Cloning of NS5B PCR samples amplified from infected patients into pSC_1b_NS5B_5'AsiSI_3'SacII and pSC_1b_delNS5B_5'AsiSI_3'SacII

[0064] Figure 2 provides a schematic representation of the experimental design for cloning patient-derived NS5B PCR samples into a replicon shuttle vector and detecting the resulting replicon. Briefly, the DNA sequence encoding the NS5B protein was generated by PCR directly from plasma of HCV-infected patients or from DNA amplified from plasma, using denaturing primers or patient-specific primers in which the sense strand primer contained the AsiSI restriction site sequence , while the antisense strand primer contains a SacII restriction site sequence. The sense strand AsiSI primers used included:

[0065] 5'-GGAGGCTAGTGAGGAC GCGATCGC TTGCTCAATGTCCTA-3'

[0066] (SEQ ID NO: 9)

[0067] 5'-GGAGGCTAGTGAGGAC GCGATCGC TTGCTCAATGTCTTA-3'

[0068] (SEQ ID NO: 10)

[0069] 5'-GGAGGCTAGTGAGGAC GCGATCGC TTGCTCRATGTCCTA-3'

[0070] (SEQ ID...

Embodiment 3

[0104]Cloning of NS5B PCR samples amplified from infected patients into pSC_1b_NS5B_5'AsiSI_3'RsrII, pSC_1b_5'AsiSI_lacZAmC_3'RsrII, pSS-1_1a_NS5B_5'AsiSI_3'RsrII and pSS-1_1a_5'AsiSI_lacZAmC_3'RsrII

[0105] NS5B PCR samples were cloned into a vector containing RsrII at the 3' end of the NS5B gene using the procedure described in Example 2, except that patient NS5B was PCR amplified using the following antisense strand primers containing a RsrII restriction site:

[0106] 5'-GGCCTGGAGTGTTTAGCT CGGACCG TCATCGRTTGGGGAG-3'

[0107] (SEQ ID NO: 25)

[0108] 5'-GGCCTGGAGTGTTTAGCT CGGACCG TCACCGGTTGGGGAG-3'

[0109] (SEQ ID NO: 26)

[0110] 5'-GGCCTGGAGTGTTTAGCT CGGACCG TCAYCGGTTGGGGAG-3'

[0111] (SEQ ID NO: 27)

[0112] 5'-GGCCTGGAGTGTTTAGCT CGGACCG TCATCGGTTGGGAAG-3'

[0113] (SEQ ID NO: 28)

[0114] 5'-GGCCTGGAGTGTTTAGCT CGGACCG TCAACGGTTGGGAAG-3'

[0115] (SEQ ID NO: 29)

[0116] 5'-GGCCTGGAGTGTTTAGCT CGGACCG TCAACGGTTGGGGTA-3'

[0117] (SEQ ID NO: 30)

...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides for novel HCV replicon shuttle vectors useful for cloning in HCV polynucleotide sequences from samples of HCV-infected patients and testing the resulting replicons for drug susceptibility.

Description

field of invention [0001] The present invention relates to novel HCV replicon shuttle vectors which are useful for screening, testing and evaluating HCV polymerase inhibitors. Background of the invention [0002] Hepatitis C virus is a serious health problem and a major cause of chronic liver disease throughout the world (Boyer, N. et al. J. Hepatol. 2000 32:98-112). Patients infected with HCV are at risk of developing cirrhosis followed by hepatocellular carcinoma, so HCV is a leading indicator for liver transplantation. [0003] According to the World Health Organization, there are more than 200 million infected people worldwide, of whom at least 3-4 million are infected every year. Once infected, about 20% of people clear the virus, and the rest carry HCV for life. 10-20% of people with chronic infection eventually develop cirrhosis or cancer that destroys the liver. This viral disease is transmitted parenterally through contaminated blood and blood products, contamina...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/51C12Q1/68
CPCC12N2770/24243C12N2770/24222C12N15/86C07K14/005
Inventor P·S·迪特里希A·H·科萨卡S·勒波甘I·纳杰拉
Owner F HOFFMANN LA ROCHE & CO AG
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com