Method for building hepatitis B e-antigen negative mouse model
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A hepatitis B, mouse model technology, applied in the field of genetic engineering
Inactive Publication Date: 2017-06-20
CHONGQING MEDICAL UNIVERSITY +1
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However, the current research on chronic HBV infection and treatment is mainly based on e-positive animal models. So far, there is no research and report on ...
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Embodiment 1
[0038] Embodiment one, construct the mouse model of hepatitis B e antigen negative
[0039] Follow the steps below:
[0040] 1. Cloning of full-length DNA of HBV e-negative virus strain
[0041] 1. Amplify the full length of HBV DNA: extract the DNA from the serum of patient No. 11061008 (provided by the Department of Infectious Diseases, the Second Affiliated Hospital of Chongqing Medical University) diagnosed as HBeAg-negative infection, and use primers to perform PCR amplification of HBV-F / HBV-R , the primer sequence is:
[0046] 2. Sequence the PCR product obtained in step 1 to obtain its sequence, redesign the primers according to the sequencing results, delete 7 bases in the newly designed upstream and downstream primers, and change the degenerate bases of the upstream primers accordi...
Embodiment 2
[0056] Embodiment two, the detection of mouse model
Embodiment 1
[0057] Detect the experimental group, control group, and blank group mouse models constructed in Example 1:
[0058] 1. Detection of HBV-specific markers in mouse serum
[0059] Specific markers of HBV such as HBsAg, HBeAg, viral DNA, and alanine aminotransferase (ALT) were detected in the serum of experimental mice at different time points. The detection of HBsAg and HBeAg was performed using a radioimmunoassay kit; the detection of ALT was performed using a small Mouse ELISA Kit.
[0060] The real-time fluorescent quantitative PCR method was used for the detection of HBV DNA. 50 μL serum was taken for extraction of viral DNA and RNA, 2 μL total DNA was used for qPCR, and the HBV copy number was analyzed. Primers were designed and amplified in the conserved region of the HBV gene, forward primer: HBV-Q-F: 5′-CCTCTTCATCCTGCTGCT-3′; reverse primer HBV-Q-R: 5′-AACTGAAAGCCAAACAGTG-3′, the standard curve uses pEASY-HBV / HBeAg-negative HBVDNA by 5×10 3 ,5×10 4 ,5×10 5 ,5×10 6...
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Abstract
The invention discloses a method for building a hepatitis B e-antigen negative mouse model. The method comprises the following steps: 1) extracting serum DNA of a patient diagnosed with HBeAg negative HBV infection, and carrying out PCR amplification by using a primer pair HBV-F/HBV-R; 2) sequencing a PCR product, and redesigning a specific PCR amplification primer pair to the sequence for PCR amplification; 3) cloning the PCR product to a pEASY-Blunt Zero carrier; 4) extracting plasmids, carrying out enzyme digestion, recovering and purifying fragments, and carrying out cyclization and purification to obtain HBVe negative cccDNA; and 5) injecting the cccDNA into a mouse, thereby building the hepatitis B e-antigen negative mouse model. According to the invention, the hepatitis B e-antigen negative mouse model is successfully built for the first time, which is highly significant for study on the mechanism of the HBeAg negative hepatitis infection and study on medication effects.
Description
technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing a hepatitis B e antigen-negative mouse model. Background technique [0002] Viral hepatitis B is a major global health problem caused by hepatitis B virus (HBV). Chronic hepatitis B caused by HBV infection may develop into cirrhosis and hepatocellular carcinoma. Recently, it was reported that about 248 million people in the world suffer from chronic hepatitis B (CHB). The serological classification of chronic hepatitis B is very complicated. According to the level of serum e antigen, it can be divided into e antigen positive and e antigen negative chronic hepatitis B infection. In recent years, the number of HBV infections in the world has decreased, but the number of HBeAg-negative infections is increasing. However, the current response rate to antiviral therapy for e-antigen-negative patients is low, resulting in increased processing t...
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