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Production of mouse model of viral hepatitis B with high expression HBsAg

A viral hepatitis, mouse model technology, applied in the preparations, pharmaceutical formulations, recombinant DNA technology and other directions for in vivo experiments, can solve the problems of inability to cause liver inflammation, inability to directly apply HBV infection, and large differences in biological characteristics. , to achieve the effect of simple and easy production process, short production process time and easy operation

Inactive Publication Date: 2007-07-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, currently used embryonic HBV transgenic animals are in a state of immune tolerance to the main gene products of HBV and cannot cause obvious liver inflammation; however, the biological characteristics of DHBV, WHV and HBV are quite different, and the research results cannot be concluded. Directly applied to the research of HBV infection

Method used

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  • Production of mouse model of viral hepatitis B with high expression HBsAg
  • Production of mouse model of viral hepatitis B with high expression HBsAg
  • Production of mouse model of viral hepatitis B with high expression HBsAg

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: Construction of recombinant plasmid pcDNA3 / 1.1HBV

[0060] 1. Materials

[0061] The plasmid p3.6II containing 1.1 times the full gene of hepatitis B virus was kindly donated by Professor Wang Yuan from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Plasmid extraction kits and DNA recovery kits are Qiagen products. The BioPhotometer quantitative instrument is a product of Eppendorf in Germany, the automatic gel imaging system is a product of AmershamPharmacia Biotech in the United States, the ZF-BI ultraviolet spectrophotometer is manufactured by Shanghai Changming Electronic Instrument Factory, and the HZS-H constant temperature water bath oscillator is manufactured by Harbin East The product of Lian Electronic Technology Development Co., Ltd., the digital display electric heating constant temperature water bath box (HH·W21·600S) is manufactured by Shanghai Yuejin Medical Equipment Factory, and the LKB2103 electrophores...

Embodiment 2

[0072] Embodiment 2: Amplification, extraction and purification of recombinant plasmid pcDNA3 / 1.1HBV

[0073] 1. Materials

[0074] (1) Plasmid: The recombinant plasmid pcDNA3 / 1.1HBV carrying 1.1 copies of the whole HBV genome was constructed and preserved by our laboratory. The recombinant plasmid pcDNA3 / 1.1HBV was stored in Escherichia coli DH5α.

[0075] (2) Reagents: The plasmid mass extraction kit is a product of QIAGEN, Germany. The kit contains 10 pieces of QIAGEN-tip500, Buffer P1 1×110ml, Buffer P2 1×110ml, Buffer P3 1×110ml, Buffer QBT 1×110ml, Buffer QC 3×240ml, Buffer QF 1×170ml, RNase A*1× 11mg.

[0076] (3) Instruments: The gel automatic imaging system is a product of Amersham Pharmacia Biotech in the United States, the ZF-BI ultraviolet spectrophotometer is manufactured by Shanghai Changming Electronic Instrument Factory, and the HZS-H constant temperature water bath oscillator is a product of Harbin Donglian Electronic Technology The product of Development ...

Embodiment 3

[0100] Example 3: Tail vein high pressure injection to establish mouse hepatitis model

[0101] 1. Materials

[0102] (1) Animals: SPF-grade BALB / c mice, 4-6 weeks old, male, weighing 18-22 g, provided by the Experimental Animal Center of Shandong University, and the feeding conditions were in accordance with the standards for SPF-grade animals.

[0103] (2) Reagent: pcDNA3-HBV1.1 purified by QIAGEN Plasmid Mass Extraction Kit, adjusted concentration to 20-50 μg / ml; physiological saline (0.9% NaCl solution).

[0104] (3) Instruments: 1ml and 5ml syringes; 0.55mm needle.

[0105] 2. Method

[0106] (1) Tail vein high-pressure injection: first place the mouse in a high-temperature environment to expand the tail vein; anesthetize the mouse with ether before injection, and observe the reflex of the mouse; within 5 seconds, inject 1.4-2.0ml of liquid into the small vein through the tail vein at a uniform speed. In mice, the mice were placed at 25-28°C.

[0107] (2) Blood colle...

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Abstract

A process for preparing VHB mouse model with high expression of HBsAg includes such steps as linearizing carrier pcDNA3 by EcoRV, dephosphorylating by alkaline phosphatase of ox's small intestine, recombining the clonal carrier p3.6II, enzyme severing by PvuII to obtain 1.1-time HBV gene fragment, linking it with linear pcDNA3, transforming colibacillus competent cell DH5 alpha, screening resistance, enzyme serving, sequencing to verify the recombinant plasmid, injecting it in the mouse body via tail vein, and verifying the model.

Description

technical field [0001] The invention relates to a preparation method of an animal model, in particular to a preparation method of a mouse model which highly expresses HBsAg and can better simulate human hepatitis B virus. Background technique [0002] Hepatitis B is an infectious disease caused by hepatitis B virus (HBV). At present, there are about 400 million patients with chronic HBV infection in the world; in China, the HBsAg carrier rate is as high as 9.75%, and there are about 30 million patients with chronic hepatitis B, of which 10% to 20% can develop liver cirrhosis, and 1% to 5% can develop liver cirrhosis. Evolved into hepatocellular carcinoma (hepatocellular.carcinoma, HCC). Hepatitis B is an immune disease, and its pathogenesis is not yet fully understood; although many in vitro and in vivo studies have been carried out with the help of cell models and animal models, due to the limitations and defects of these models in various aspects, no progress has been mad...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/52A61K49/00
Inventor 孙汶生刘玉刚张秋高立芬石永玉刘素侠朱法良郭春
Owner SHANDONG UNIV
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