Method for constructing the regulation and expression of hepatitis C virus NS3/NS4A protease

A hepatitis C virus, regulation and expression technology, applied in the field of regulation and expression protease construction, can solve the problems of high cost, inconvenient source of chimpanzee, difficult operation of chimeric mouse model, etc.

Inactive Publication Date: 2010-03-17
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

Due to the inconvenient source of the chimpanzee used in the infection animal model, and the difficulty and high cost of the chimeric mouse model, there are great limitations in application.

Method used

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  • Method for constructing the regulation and expression of hepatitis C virus NS3/NS4A protease
  • Method for constructing the regulation and expression of hepatitis C virus NS3/NS4A protease
  • Method for constructing the regulation and expression of hepatitis C virus NS3/NS4A protease

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Embodiment Construction

[0034] The present invention will be further described below in conjunction with specific examples. It should be noted that the following examples are not intended to limit the implementation scope of the present invention.

[0035] 1. Construct a transgenic vector expressing NS3 / NS4A protease. (see attached figure 1 )

[0036] attached figure 1 Middle: Laz: β-galactosidase reporter gene; P CMV : CMV promoter; tetO: tetracycline operator element; Ptetbi-1: composed of tetO sequence and P on both sides CMV Constituent bidirectional promoter; LoxP: Cre recombinase cleavage site; dox: doxycycline hydrochloride; Luc: luciferase reporter gene; pA: polyA tail.

[0037] (1) On the eukaryotic expression vector PBI-3 containing the bidirectional promoter Ptetbi-1, the transgenic eukaryotic expression vector PBI-3-Luc- pA. (see attached figure 2 )

[0038] attached figure 2 Middle: 1: DNA molecular weight marker - DL2000Marker, 2: After PBI-3-Luc-pA is digested with PstI and ...

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Abstract

The invention relates to a method for constructing the regulation and expression of a protease, in particular to a method for constructing the regulation and expression of a hepatitis C virus NS3/NS4A protease; the genome of transgenic animals contains a transgene DNA of a coding hepatitis C virus NS3/NS4A, moreover a regulating gene sequence and a report gene sequence are added before a purpose gene HCV NS3/4A DNA so as to be capable of expressing the active NS3/NS4A protease on the condition of a regulating factor. The method for constructing the regulation and expression of the hepatitis Cvirus NS3/NS4A protease comprises the following contents: (1) constructing a eukaryotic expression vector which contains regulating sequences such as LoxP locus, Luciferase, and polyA and the like, is dually regulated by Tet and Cre/LoxP systems, and expresses the HCV NS3/4A protease; and (2) obtaining an NS3/4A mouse and a transgenic heterozygous mouse with the genetic background basically similar to the wild type mouse. The HCV NS3/NS4A transgenic mouse model obtained can be used for the hybridization with a double-transgenic mouse, and further obtains a three-transgenic mouse and is applied to the HCV pathogenesis research and drug screening as well as evaluation.

Description

technical field [0001] The invention relates to a construction method for regulating and expressing protease, in particular to a construction method for regulating and expressing hepatitis C virus NS3 / NS4A protease and its application. Background technique [0002] Hepatitis C virus is the main pathogen causing chronic liver disease, seriously endangering human health, and has become a global and important public health problem. So far, there is still a lack of effective antiviral treatment measures and effective vaccines. [0003] At present, the most effective treatment for HCV is only the combined use of interferon and ribavirin, the effect varies greatly among different genotypes, the curative effect is less than 40%, and the side effects are very serious. With the clinical application of protease and reverse transcriptase inhibitors in the treatment of HIV and the good curative effect achieved, in recent years, enzymes (such as serine proteases) that play a key role in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027C12N9/50C12Q1/37C12R1/93
Inventor 尹文赵亚孙梦宁吕欣雷迎峰杨敬康健姚敏薛采芳
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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