Method for constructing chronic transfection model of hepatitis B virus

A technology of hepatitis B virus and construction method, which is applied in the field of construction of hepatitis B virus chronic transfection model, can solve the problems of high price, low chronicity rate, short duration, etc., and achieve low cost and high chronicity model rate Enhanced, accessible results

Active Publication Date: 2015-06-03
SHANGHAI PUBLIC HEALTH CLINICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current treatment measures for the disease include nucleoside drugs such as lamivudine, adefovir dipivoxil, etc., but the drugs can only inhibit virus replication to a certain extent and easily cause virus drug-resistant mutations, while conventional interferon -α (IFN-α) and pegylated IFN-α are only effective in some patients (less than 30% response rate)
[0004] The prior art has been published, and the animal infection model established for HBV research, however, the existing HBV infection animal models have great limitations
For example, chimpanzees are endangered animals and are expensive and involve ethical issues, so their use is strictly restricted; in fact, experimental research on non-human primates has been banned in Europe, and the NIH in the United States does not support it; although tree shrews can infect HBV, but cannot continue to replicate to cause chronic infection, there is a certain gap with clinical practice; the infection model established by other hepadophilic viruses such as duck hepatitis B virus also has different gene transcription regulation mechanisms, and the immune system of poultry and humans is far different; Commonly used HBV mouse models, such as transgenic mouse models, HBV-infected human liver chimeric mouse models, and acute/chronic HBV transfected mouse models, have their own characte

Method used

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  • Method for constructing chronic transfection model of hepatitis B virus
  • Method for constructing chronic transfection model of hepatitis B virus
  • Method for constructing chronic transfection model of hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1) Prepare pAAV / HBV1.2 or 1.3 plasmid

[0034] Use Escherichia coli to amplify the pAAV / HBV1.2 or 1.3 plasmid, and obtain the required plasmid through kit extraction, and determine the concentration and purity of the plasmid;

[0035] 2. Modeling by high pressure hydrodynamic method

[0036] Take 10 male C3H / HeN mice of SPF grade 4-8 weeks old, dissolve 5 μg pAAV / HBV1.2 or 1.3 plasmid in 8% (volume / mouse body weight) PBS solution, and quickly transfect the experimental mice by Rapid transfection to mice within 3-10 seconds via tail vein; C57BL / 6 mice of the same age were used as controls;

[0037] 3. Monitor the persistence of virological indicators in mice, and evaluate the success of chronic modeling,

[0038]On the third day after the plasmid was transfected, the blood of the mice was collected to separate the serum, and whether the hepatitis B surface antigen (HBsAg) was detected in the serum by ELISA was positive to determine whether the transfection was successf...

Embodiment 2

[0041] Use Escherichia coli to amplify the pAAV / HBV1.2 or 1.3 plasmid, and obtain the required plasmid through kit extraction, measure the concentration and purity of the plasmid, and confirm that there is no endotoxin, protein, and genomic DNA contamination;

[0042] .Take 10 male C3H / HeN mice of SPF grade 4-8 weeks old (with C57BL / 6 mice of the same age and sex as the control), dissolve 10 μg of pAAV / HBV1.2 or 1.3 plasmid in 12% ( Volume / mouse body weight) in PBS solution, quickly transfected into mice within 3-10 seconds through the tail vein,

[0043] On the third day after the plasmid injection, 50 μl of blood from the fundus venous plexus of the mouse was collected, and whether HBsAg was detected in the serum by ELISA method was used to determine whether the high-pressure hydrodynamic tail vein transfection of the plasmid was successful or not.

[0044] For the mice with successful plasmid transfection, blood from the fundus venous plexus was regularly collected every we...

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a method for constructing a chronic transfection model of a hepatitis B virus. The method disclosed by the invention comprises the following steps: transfecting HBV (hepatitis B virus) plasmids into a model-preparing experimental mouse (preferably adopting male C3H/HeN of 4-8-week old), and constructing the chronic transfection model of the HBV. The chronic transfection model of the hepatitis B virus constructed by the method can be used for researching specific action mechanism of single or multiple genes in an HBV pathogenic process; transgenic modification of the mouse is not needed; the method is free of limitations such as immunologic tolerance, easy to obtain, and low in cost; the problem of long-term shortage of an animal model suitable for the HBV in related technical fields can be solved; and a good animal model is provided for further research on life history and chronic mechanism of the HBV and screening of vaccines and new medicines.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a chronic transfection model of hepatitis B virus, in particular to a method for constructing a chronic transfection model of hepatitis B virus. Background technique [0002] As far as hepatitis B virus (hepatitis B virus, HBV) is concerned, there are about 2 billion infected people worldwide, of which 350 million people become chronic hepatitis B virus carriers, and there are about 90 million hepatitis B virus carriers in China. According to reports, chronic infection of hepatitis B virus often leads to serious diseases such as liver cirrhosis and liver cancer; the direct medical expenses for hepatitis and liver disease in China are as high as more than 100 billion yuan every year. The current treatment measures for the disease include nucleoside drugs such as lamivudine, adefovir dipivoxil, etc., but the drugs can only inhibit virus replication to a certain extent and easily cause virus...

Claims

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Application Information

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IPC IPC(8): C12N15/864A01K67/027
Inventor 周晓辉彭秀华任晓楠陈丽香徐春华方钟
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
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