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Method for constructing hepatitis B virus (HBV) infected mouse model

A hepatitis B virus, mouse model technology, applied in the biological field, can solve the problems of complex process, toxicity of adenovirus, limited application, etc., and achieve the effects of simple construction process, easy technical means and low construction cost

Active Publication Date: 2014-12-17
张欣欣 +6
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Problems solved by technology

[0003] (1) The transgenic mouse model is to insert the HBV genome into the mouse genome through transgenic technology, which can produce HBV virus particles similar to those in humans and is infectious. It can be used for in vivo research on the pathogenic mechanism of HBV, and can also be used for anti-cancer The related research on viral drugs to inhibit virus replication has its inherent deficiencies: first, transgenic technology is relatively complex and costly; second, due to unknown reasons, the HBV gene integrated into the mouse genome cannot produce cccDNA (in humans cccDNA is the natural transcription template for HBV replication); in addition, due to the transgenic relationship, the immune system of mice recognizes the virus as its own substance, and it needs to be transferred to specific T lymphocytes to carry out relevant immune research, which limits The range of research applications of the model
[0004] (2) The mouse model of viral vector transfection is to transfect the adenoviral vector carrying the HBV genome into the mouse body. Through the adenoviral vector, the HBV genome can be effectively transfected into the mouse liver and replicated, and can produce mild Moderate liver inflammation and elevated serum alanine aminotransferase (ALT) can be used to study the immune-mediated virus clearance mechanism in vivo. It also has its own shortcomings. Adenovirus itself is toxic and can induce the body's immune response. Response limits its application
[0005] (3) The chimeric mouse model first needs to be irradiated to inactivate its bone marrow cells, recombined with bone marrow cells of severe combined immunodeficiency mice (SCID mice) as a pretreatment, and then transplanted into human liver tissue fragments that have been infected with HBV in vitro to construct Chimera mouse model, in addition to constructing a chimera model through immunodeficiency transgenic mice, such as uPA\SCID mice, since it carries human liver cells, this model can also be used for superinfection with other human hepatotropic viruses However, the construction of chimeric mouse model is costly and complicated, and because it is a severe immunodeficiency mouse, its application in vaccines and adaptive immune response mechanisms is limited

Method used

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  • Method for constructing hepatitis B virus (HBV) infected mouse model
  • Method for constructing hepatitis B virus (HBV) infected mouse model
  • Method for constructing hepatitis B virus (HBV) infected mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Identification of Mouse Tlr2 Genotypes:

[0049] (1) Extract the genomic DNA of the experimental mouse: cut the tail of the mouse about 0.8 cm, lyse it with 50 μl proteinase K solution (10 mg / mL), put it in a water bath at 56 °C overnight, and centrifuge the next day (12000 rpm, room temperature for 10 Minutes), add 0.5ml of 70% ethanol, inhale several times until a flocculent precipitate appears, centrifuge at 13200 rpm for 3 minutes at room temperature, discard the supernatant and dry it, add 200μl ddH 2 Mouse genomic DNA was obtained by dissolving in O and stored at 4°C.

[0050] (2) Identification of mouse genotype by PCR and gel electrophoresis experiments:

[0051] PCR system preparation (50μl):

[0052]

[0053] Table 1

[0054]

[0055] The PCR amplification program is shown in Table 2:

[0056] Table 2

[0057]

[0058]

[0059] The products obtained by PCR were subjected to agarose gel electrophoresis: the Tlr2 genotype of the mouse was identi...

Embodiment 2

[0061] Sampling for detection of virological indicators:

[0062] Tail vein high pressure injection: within 5 to 10 seconds, inject 2 ml of Ringer solution in which the corresponding plasmid was dissolved through the tail vein of the mouse within 5 to 10 seconds, and each mouse was injected once in total. The experimental mice in the R subgroup of each group were only injected with 2ml of Ringer solution without any plasmid, each mouse in the K subgroup was injected with 20 μg of empty vector plasmid, and each mouse in the W subgroup was injected with 20 μg of Wild-type HBV plasmid, M subgroup was injected with 20 μg of mutant HBV plasmid per mouse.

[0063] Peripheral blood collection and processing: Peripheral blood was collected through the infraorbital venous plexus of mice, and about 200 μl of whole blood was collected each time. The blood collection time points were blood collection before tail vein hyperbaric injection (day0), and 3 days after tail vein hyperbaric injec...

Embodiment 3

[0065] Serum ALT detection: Take 50 μl of mouse serum at each time point of day0, day3, day7, day10, day14, day17, day21 and day28 of each group, add 0.9% normal saline (NS) at a ratio of 1:1, and mix well Afterwards, the ALT level was detected with a Beckman Coulter automatic biochemical analyzer, and the unit of the measured value was IU / L. The changes of serum ALT levels in experimental mice were as follows: Figure 4 Shown, prove that the acute HBV infection mouse model (comprising homozygous and heterozygous Tlr2 gene deficient mouse) that the present invention builds by experiment than the HBV infection model that existing C57BL / 6 (B6) mouse builds, has more A high ALT level can better simulate the biochemical manifestations of hepatitis caused by HBV infection. Additionally, if figure 1 As shown, compared with the data after three weeks of injecting the plasmid, the mouse model of the present invention (CW, ZW, CM, ZM group mice) has significantly increased serum ALT ...

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Abstract

The invention relates to the technical field of biology and in particular relates to a method for constructing a hepatitis B virus (HBV) infected mouse model and application of the HBV infected mouse model. The invention provides a method for constructing a high-ALT (alanine transaminase) HBV infected mouse model. The method for constructing the HBV infected mouse model comprises the step of medicating HBV genome DNA plasmid to a Tlr2 gene-deficient mouse, so that the HBV infected mouse model is constructed. The mouse model provided by the invention is relatively simple to construct, relatively easy in technological means and low in construction cost; besides, the adopted HBV plasmid vector is non-toxic, can not induce immune response of the mouse and also can avoid serious immunodeficiency in a chimera mouse model, and the obtained HBV infected model has higher blood serum ALT level and obvious liver inflammation expression.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for constructing a mouse model of hepatitis B virus infection and its use, and further relates to the mouse obtained by the method for constructing a mouse model of hepatitis B virus infection. Models and their uses. The animal model constructed by the invention can be used for in vivo experiments on the mechanism of HBV acute infection, and has good application value in the research of HBV infection and pathogenesis. Background technique [0002] Existing mouse models for HBV infection in vivo experiments are divided into transgenic mouse models, viral vector transfection mouse models, chimeric mouse models and tail vein hypertensive injection mouse models according to their construction methods. There are some problems, specifically: [0003] (1) The transgenic mouse model is to insert the HBV genome into the mouse genome through transgenic technology, which can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027A61K49/00
Inventor 张欣欣刘晶谷雷雷韩悦杨慧娟谢幼华沈忠良
Owner 张欣欣
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