DNA (Deoxyribonucleic acid) segment and applications thereof in constructing HCV (hepatitis C virus) whole-genome expression animal model

A whole genome and fragment technology, applied in DNA/RNA fragments, recombinant DNA technology, cells modified by introducing foreign genetic material, etc., can solve the problems of not reflecting antiviral effect, immunopathological damage, etc.

Inactive Publication Date: 2013-07-10
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since this model mouse is in a state of immune tolerance or immunodeficiency, it cannot reflect the antiviral effect of the host's immu

Method used

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  • DNA (Deoxyribonucleic acid) segment and applications thereof in constructing HCV (hepatitis C virus) whole-genome expression animal model
  • DNA (Deoxyribonucleic acid) segment and applications thereof in constructing HCV (hepatitis C virus) whole-genome expression animal model
  • DNA (Deoxyribonucleic acid) segment and applications thereof in constructing HCV (hepatitis C virus) whole-genome expression animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, construction contains the recombinant vector of DNA segment of the present invention

[0037] 1. Using the method of overlapping PCR in the plasmid pJFH1 (document: Production of Infectious Hepatitis C Virus in Tissue Culture from A Cloned Viral Genome. Nat. Med, 2005; 11 (7): 791-796. The public can obtain it from the Chinese People's Liberation Army Military Acquired by the Institute of Microbial Epidemiology, Academy of Medical Sciences) the HCV whole genome gene (shown in the 486th-10220th position of the sequence listing sequence 1) downstream forwardly connects the hepatitis D virus (HDV) ribozyme gene sequence (the sequence listing sequence 1 position 10221-10308), to obtain recombinant vector A;

[0038] 2. In order to avoid the potential impact of the prokaryotic promoter on the expression of HCV in the transgenic animal, the prokaryotic promoter T7 upstream of the HCV whole genome gene in the recombinant vector A obtained in step 1 was removed to...

Embodiment 2

[0043] Embodiment 2, utilize the transgenic animal prepared and identification of the DNA fragment of the present invention

[0044] The recombinant vector pTRE2-HCV-ribo-delete7 obtained in Example 1 was used with XbaI (the recognition sequence is T^CTAGA, located at the 10308-10313th position of sequence listing sequence 1) and PvuI (the recognition sequence is CGAT^CG, located in the sequence listing The 12919-12924 position of Sequence 1) was double-enzymatically digested, and the 10924bp large fragment was recovered, and the prokaryotic DNA of C57BL / 6×CBA F1 mouse fertilized eggs (Guangzhou Saiye Biotechnology Co., Ltd.) was microinjected, and the injected eggs were respectively Transplanted into the fallopian tubes of pseudopregnant female mice, cut the tail of about 0.5 cm from the second week after birth, and extracted genomic DNA for PCR identification. The primer sequences are as follows:

[0045] HCV-F: 5'-CTTGTTGTGCCCTACGGATTG-3';

[0046] HCV-R: 5'-TGAAGGTGGAGAAG...

Embodiment 3

[0049] Example 3, Preparation of Tet-on-HCV Double Transgenic Mice and Identification of Gene Integration

[0050] The transgenic mouse "albumin-rtTA transgenic" that specifically expresses rtTA in the liver tissue regulated by the mouse albumin promoter (document: Xiao-Jun Zhou et al. Over-expression of uPA increases risk of liver injury in pAAV-HBV transfected mice.World J Gastroenterol 2012,18:1892-1902. The public can obtain it from the Institute of Microbial Epidemiology, Academy of Military Medical Sciences of the Chinese People’s Liberation Army) and TRE2-HCV transgenic mice, PCR identification of both integrated rtTA and HCV genome-wide genes Double transgenic mice. The identification primers and PCR conditions of HCV whole genome gene integration are the same as in Example 2, and the integration identification primers of rtTA are as follows:

[0051] rtTA-F:5'-GTGCAGCTTGGCTTGAACTCGTTC-3';

[0052] rtTA-R:5'-GAGTATGGTGCCTATCTAACATCTC-3';

[0053] The PCR reaction sy...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) segment and applications thereof in constructing an HCV (hepatitis C virus) whole-genome expression animal model. The nucleotide sequence of the DNA segment comprises the following sequentially series connected sequences: an operator gene sequence of bacteria tetracycline drug-resistance operon, an immediate early promoter gene sequence of human macrophage, a gene sequence of HCV whole genome, and a gene sequence of ribozyme shearing the HCV whole genome activity on DNA and/or RNA (ribonucleic acid) level. The DNA segment can be used for constructing a tetracycline or tetracycline derivative-induced HCV whole genome-expressed transgenic animal model. The immunity of the animal model is normal, thereby being capable of producing specific anti-virus immunoreaction aiming at the expression of the HCV, and further being effectively applied to the research on the immunopathogenesis damage mechanism of drug evaluation and virus infection of the HCV vaccine.

Description

technical field [0001] The present invention relates to a DNA fragment and its application in constructing HCV full gene expression animal model. Background technique [0002] Hepatitis C virus (HCV) is a hepatotropic single-stranded positive-sense RNA virus with a genome size of about 9.6 kb, including an ORF encoding polyprotein and non-coding regions on both sides. At present, there are about 180 million HCV-infected patients in the world, and about 20% of them will develop into liver cirrhosis and further develop into liver cancer, thus seriously threatening human health. HCV is highly species-specific and can only infect humans and chimpanzees, but cannot naturally infect mice, thus seriously hindering the research on HCV replication and its pathogenic mechanism, as well as the development and evaluation of vaccines and antiviral drugs. The establishment of new HCV small animal models is of great significance for the study of HCV host susceptibility and replication mec...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N7/01A01K67/027
Inventor 周育森孙世惠周小军刘晨风赵光宇曾扬
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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