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Reporter gene labeled-mouse model for monitoring function of HBV specific CTLs in vivo and construction method and application thereof

A technology of reporter gene and construction method, which is applied in the mouse model of reporter gene marker for monitoring the function of HBV-specific CTLs in vivo and its construction and application field, which can solve errors, only reflect the past immune response effect, low sensitivity, etc. question

Active Publication Date: 2011-02-23
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

Although they are widely recognized parameters for measuring antiviral immunity, they have the disadvantages of low sensitivity, sample error, and only reflect the effect of past immune responses.

Method used

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  • Reporter gene labeled-mouse model for monitoring function of HBV specific CTLs in vivo and construction method and application thereof
  • Reporter gene labeled-mouse model for monitoring function of HBV specific CTLs in vivo and construction method and application thereof
  • Reporter gene labeled-mouse model for monitoring function of HBV specific CTLs in vivo and construction method and application thereof

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Embodiment 1

[0044] Embodiment 1, the construction of the mouse model of the reporter gene mark of monitoring HBV-specific CTLs function in vivo

[0045] Use the method of the present invention to construct the mouse model of the reporter gene marker of monitoring HBV-specific CTLs function in vivo, and specific method comprises the following steps:

[0046] 1. Construction of recombinant plasmid pGL3-CP-Fluc-HBV1.2 capable of expressing reporter gene and HBV antigen in vivo

[0047] HBV 1.2-fold genome (subtype adw, gene type A) recombinant expression vector pAAV / HBV1.2 containing AAV (Huang LR, Wu HL, Chen PJ and Chen DS. Proc Natl Acad Sci U S A. 2006, 103: 17862-17867 .) including HBV genome 1400-3182bp+1-1987bp and two inverted repeats (ITR). Take advantage of high-fidelity PrimeSTAR TM HS DNA polymerase was amplified by PCR to obtain a complete 1.2-fold HBV gene fragment containing ITR, which was reversely inserted into the eukaryotic expression vector pGL3-CP-Fluc (Juan Du, YongZh...

Embodiment 2

[0081]Example 2, Reporter Gene Marked HBV Stable Expression Mouse Model Used for Detection of HBV-specific CTLs Function Evoked by Vaccine

[0082] 1. Construction of recombinant eukaryotic expression vector pVAX1-HBc

[0083] 1.1 The primers for the expression gene HBc of HBV core antigen were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The primer sequences are as follows:

[0084] PC (upstream primer): 5'-CGCGGATCCATGGACATTGACCCTTTAT-3';

[0085] PD (downstream primer): 5'-CCGGAATTCCTAACATTGAGATTCCCG-3'.

[0086] 1.2 Obtaining HBc fragments

[0087] Using pAAV / HBV1.2 as a template, using Taq DNA polymerase, using PC and PD primers to amplify the gene fragment of HBc with EcoRI and BamHI restriction sites, after the amplification, perform 1% agarose gel electrophoresis, The amplified product was recovered with a gel recovery purification kit (purchased from TaKaRa Company). Among them, the PCR reaction system is: pAAV / HBV1.23μl, Ex Taq 0.5μl, upstream and down...

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Abstract

The invention discloses a reporter gene labeled-mouse model for monitoring the function of HBV specific CTLs in vivo and a construction method and application thereof. The construction method comprises the following steps of: 1) constructing a vector, namely constructing a recombinant expression vector comprising an HBV promoter, a reporter gene and an HBV genome; and 2) transfecting the constructed recombinant expression vector into a mouse body so as to prepare the reporter gene labeled-mouse model for monitoring the function of HBV specific CTLs in vivo. A visual mouse model, which is simple, practicable, stable and repeatable, is constructed, and the model can be used for monitoring destructive and nondestructive functions of HBV specific CTLs. In addition, the mouse model is constructed according to an adult mouse by a hydrodynamic transfection method, does not need any special instrument, and has the advantages of short research cycle, low cost, specific, long-term and stable expression of a target gene in liver, high expression, wide application prospect and the like.

Description

technical field [0001] The invention belongs to the animal model and its construction method in the field of biotechnology, in particular to a reporter gene-marked mouse model for monitoring the function of HBV-specific CTLs in vivo and its construction method and its ability to monitor the lethality and Applications in non-lethal functions. Background technique [0002] Hepatitis B virus (Hepatitis B Virus, HBV) infection seriously affects human health, it can cause acute and chronic viral hepatitis. After acute infection, 5-10% of adults develop persistent infection, which can eventually lead to chronic hepatitis, cirrhosis, and liver cancer. At present, there are about 350 million chronically infected patients with hepatitis B virus in the world. Although the existing vaccines can effectively prevent hepatitis B virus, they cannot treat patients who have already been infected. [0003] Existing studies have shown that the body's specific cellular immune response against...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/66C12Q1/68A61K49/00C12Q1/70A01K67/027G01N33/569G01N21/64C12N15/85
Inventor 詹林盛杜娟梁声强
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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