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Method for constructing experimental animal model carrying hepatitis B virus for a long time

A hepatitis B virus, laboratory animal technology, applied in the field of genetic engineering, can solve problems such as inability to use therapeutic research, high cost of chimpanzee models, and limited application value.

Inactive Publication Date: 2019-04-19
CHONGQING ACADEMY OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chimpanzee models are expensive and limited by ethics; woodchucks and tree shrews show transient acute hepatitis; although the pathological process and prognosis of duck hepatitis are similar to those of human hepatitis B, it is now widely used It is an animal model for human hepatitis B research, but duck is a duck hepatitis virus, which is far from human hepatitis B virus, thus limiting its application value
Therefore, the existing models have certain limitations in the research on the treatment of hepatitis B, especially making them unable to be used in the research on the treatment of CHB.

Method used

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  • Method for constructing experimental animal model carrying hepatitis B virus for a long time
  • Method for constructing experimental animal model carrying hepatitis B virus for a long time
  • Method for constructing experimental animal model carrying hepatitis B virus for a long time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. HBV virus genome cloning

[0034] The serum DNA samples of hepatitis B e antigen-positive patients were provided by the Second Affiliated Hospital of Chongqing Medical University, and the patient number is 11061012. Use Phusion polymerase (High-Fidelity DNAPolymerases&MasterMixes), serum DNA template, front primer HBVF: 5'-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3', rear primer HBVR: 5'-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGT Full length sequence. The PCR amplification system is: 5×GC Buffer 10μL, 2.5mM dNTPs 4μL, 5μM front primer HBVF 1μL, 5μM rear primer HBVF 1μL, Puusion enzyme 0.5μL, serum DNA template 2μL, water supplement 50μL.

[0035] PCR amplification conditions are: (1) 98°C, 30s; (2) 98°C, 10s, 65°C, 20s, 72°C, 1min45s; this step is cycled 25 times; (3) 72°C, 10min; (4)4 Store at ℃.

[0036] Subsequently, HBV was cloned into pEASY-Blunt Zero vector by PCR amplification product to obtain pEASY-HBV positive plasmid. The specific steps are: add 3 μL PCR amplifica...

Embodiment 2

[0042] 1. Detection of serum markers HBsAg and HBeAg in mice

[0043] The blood samples taken at the set time points in Example 1 were used to detect the content of HBV serum markers HBsAg and HBeAg in the mouse serum by radioimmunoassay (using a radioimmunoassay identification kit).

[0044] by figure 1 It can be seen that the experimental group can detect HBsAg and HBeAg from the first week after injection of HBV cccDNA. figure 1 A. The HBsAg content showed a downward trend in 1-12 weeks. After the 12th week, the HBsAg content stabilized and lasted until the 26th week. figure 1 B. The content of HBeAg showed a downward trend in the second week, increased slightly from the second week to the 16th week, and then showed a downward trend, which continued until the 26th week. The contents of HBsAg and HBeAg were significantly higher than the blank control group. Prove that the serum markers of HBV can continue to express in mice for 26 weeks.

[0045] 2. The expression of HBsAg and HBc...

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Abstract

The invention discloses a method for constructing an experimental animal model carrying hepatitis B virus for a long term, which comprises the following steps of: 1) extracting serum virus DNA of a hepatitis B patient, and performing PCR amplification on HBVF / HBVR by using a primer pair; 2) cloning a PCR product into a pEASY-HBV vector, and selecting a positive clone; 3) extracting plasmid of thepositive clone, cutting the extracted plasmid with BspQI endonuclease, recovering enzyme-cut sections and purifying, cyclizing with T4 ligase and purifying to obtain HBV cccDNA; 4) injecting the cccDNA into a mouse to construct the hepatitis B e antigen negative mouse model. The method successfully constructs the CBA / CaJ mouse model with immune activity which can effectively simulate HBV chronic infection through a simple, rapid and convenient method, and the expression of HBsAg, HBeAg and HBcAg can be up to 26 weeks.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a method for constructing an experimental animal model carrying hepatitis B virus for a long time. Background technique [0002] Hepatitis B virus is caused by hepatitis B virus (HBV) infection. The disease is a serious global public health problem and can cause a variety of clinical symptoms, including chronic hepatitis B (CHB), liver Sclerosis (LC) and hepatocellular carcinoma (HCC), etc. Among them, for chronic hepatitis B, the hepatitis B virus test is positive, the course of the disease is more than half a year or the date of onset is not clear, and clinical manifestations of chronic hepatitis. According to the latest reports, about 248 million people in the world suffer from chronic hepatitis B. HBV is not easy to eliminate in the patient's body, which leads to a higher risk of the virus developing into HCC. Animal models based on HBV play a very important role i...

Claims

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Application Information

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IPC IPC(8): C12N15/867A01K67/027
CPCA01K67/0275C12N15/86A01K2227/105A01K2267/0337C12N2730/10143
Inventor 张华堂赖国旗唐雨微刘阳赵中华曹敏向钦吕小琴伍悦
Owner CHONGQING ACADEMY OF SCI & TECH
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