HCV transgene mouse model and its construction method and application

A technology of transgenic mice and models, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of low cost, short research period and overcoming systematic errors

Inactive Publication Date: 2008-08-13
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no practical and convenient small animal model that can stably evaluate the in vivo effects of anti-HCV IRES drugs

Method used

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  • HCV transgene mouse model and its construction method and application
  • HCV transgene mouse model and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, construction of bicistronic expression plasmid

[0023] (1) Construction of pCI-CMV-Rluc-HCV IRES-Fluc

[0024] like figure 1 As shown, the pRluc-CMV vector (Promega product) was digested with NheI and XbaI to obtain the Rluc gene sequence, which was connected to the pCI-neo expression vector (Promega product) that was also digested with NheI and XbaI to obtain pCI-Rluc Expression vector. Using the pHCV-neo4 vector (Chinese Journal of Microbiology and Immunology, 1999, 19(1): 17-20) (a fusion gene containing HCV IRES and firefly luciferase gene) as a template, it was prepared by Shanghai Sangon Bioengineering Technology Co., Ltd. Synthetic primers IRES-Fluc1: 5`-acgcgtcgaccccaagcttgccagcccc-3`, IRES-Fluc2: 5`-ataagaatgcggccgcagaattacacggcgatctttc-3`, the upstream and downstream primers contain SalI and NotI restriction sites respectively, and the HCV IRES-Fluc fusion gene obtained by PCR amplification The fragment was ligated with the pCI-Rluc expressio...

Embodiment 2

[0029] Example 2. The pCI-CMV-Rluc-HCV IRES-Fluc plasmid and the pCI-hAAT-Rluc-HCV IRES-Fluc plasmid were respectively hydrodynamically transfected into mice to construct an HCV transgenic mouse model

[0030]Inject a large volume (1.5-2ml) containing pCI-CMV-Rluc-HCV IRES-Fluc or pCI-hAAT-Rluc-HCV IRES-Fluc plasmid DNA (5-10 μg) in normal saline solution, and the control group was only injected with the same volume of normal saline. The mice were sacrificed at different time points 12h, 24h, 48h, 72h, 1 week, 2 weeks, and 4 weeks after injection, and the liver was prepared into a single cell suspension. The dual luciferase assay kit (Dual Luciferase Assay System, Promega) was used to detect the luciferase activity at different time points, and the recommended operation steps were followed. The results showed that using the bicistronic expression plasmid with CMV as the promoter, the target gene luciferase was transiently expressed in the mouse liver after hydrodynamic transf...

Embodiment 3

[0031] Embodiment 3, the application of HCV transgenic mouse model

[0032] Inject a large volume (1.5ml) of pCI-hAAT-Rluc-HCV IRES-Fluc plasmid into the tail vein of mice (Kunming rats, 6-8 weeks old, 18-20g) within a short period of time (5s) by hydrodynamic transfection DNA (10 μg) was the control group, and the experimental group was hydrodynamically transfected with pCI-hAAT-Rluc-HCV IRES-Fluc plasmid DNA (10 μg) and 40 μg siRNA or 40 μg DNAzyme (DNAzyme: 5'-ATGGTGCAGGCTAGCTACAACGAGGTCTACG-3', provided by Shanghai Shenggong Synthesized by Bioengineering Technology Co., Ltd.; siRNA sequence: siRNA(sense) 5'-UUGUAGACCGUGCACCAUGAGC-3', siRNA(antisense) 5'-UUGCUCAUGGUGCACGGUCUAC-3'. For the preparation method, please refer to the literature: Journal of the Academy of Military Medical Sciences. 2004, 28(2 ): 163-165) of normal saline solution, siRNA and DNAzyme are small interfering RNAs and deoxyribozymes targeting 328-347 nucleotides of HCVIRES. The results showed that 24h ...

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Abstract

A HCV transgenic mouse model is prepared through configuring the bicistronic expression carrier including sea pensy leuciferinase gene, glowworm leuciferinase gene and the HCV IRES sequence between them, and transfecting it into mouse body to obtain said model. It can be used for screening and evaluating the HCV-resistant medicines using HCV IRES as target, such as antisense oligonucleotide, specific ribozyme, siRNA, etc, and researching the affection of host factors to HCV IRES.

Description

technical field [0001] The invention relates to an HCV transgenic mouse model and its construction method and application. Background technique [0002] For a long time, the research of hepatitis C has been a major problem: the lack of small animal infection models has hindered the research process of HCV pathogenic mechanism, antiviral drug screening and vaccine research. Therefore, exploring the HCV infection model or HCV gene expression model with practical application value is a major topic in the research on the prevention and treatment of hepatitis C. [0003] The main target of antiviral therapy research on the HCV genome is the internal ribosome entry site (IRES) located in the 5' non-coding region (NCR) of the genome that mediates and controls the translation of the entire HCV genome. When drugs designed against the highly conserved region of the viral genome, IRES, are used in vivo, the possibility of virus mutants will also be greatly reduced. Therefore, IRES is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/85C12N15/51A01K67/027
Inventor 詹林盛王全立饶林彭剑淳
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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