Expression vector containing regulatably expressible cre recombinase gene and reporter gene LacZ, construction method and application thereof

Abstract

Belonging to the field of biotechnologies, the invention discloses an expression vector containing regulatably expressible cre recombinase gene and reporter gene LacZ, a construction method and application thereof. The expression vector provided by the invention is pBI-3-LacZ-cre, and the expression vector contains the lacZ reporter gene, the Cre recombinase gene and a bidirectional promoter tetO element. According to the invention, the expression vector pBI-3-LacZ-cre is introduced into a mouse fallopian tube to conduct pregnancy so as to obtain a transgenic mouse. The transgenic mouse is hybridized with two applied model animals, i.e. an rTALAP-1 mouse able to induce liver specific expression of rtTA molecules and an HBV Tg (Transgenic) mouse able to regulatably express HBV genome, and then after repeated mating, a liver inducibly expressible Tet-on and Cre / loxP combined dual-control strategy triple-transgenic mouse new model can be obtained finally. The expression vector can be effectively applied to screening and evaluation of HBV (hepatitis B virus) inhibitors or drugs, research of HBV pathogenic mechanism, host immune response after HBV infection and other key issues.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a eukaryotic expression vector and a construction method thereof, in particular to an expression vector containing a regulated and expressible cre recombinase gene and a reporter gene LacZ, a construction method and an application thereof. Background technique [0002] Hepatitis B virus (HBV) infection is still a global public health problem, and it is estimated that there are as many as 350 million HBV carriers worldwide. my country is a high-prevalence area of ​​hepatitis B, the HBV carrier rate of the population is about 10%, and some patients with chronic hepatitis can develop into liver cirrhosis or liver cancer. [0003] The pathogenic mechanism of HBV is not yet fully understood. After HBV invades the body, it mainly replicates in the liver cells to produce complete virus particles and secretes or expresses viral antigen components such as surface antigen (HBsAg), e antigen (HB...

AI Technical Summary

Problems solved by technology

However, the above-mentioned animal models also have obvious shortcomings in practical application: ① they are large in size, inconvenient to raise and operate in the laboratory, and are usually not easy to obtain; ② they are all outbred animals, with unclear genetic and immunological background; ③ different The genome structure and other aspects of this animal hepatitis virus are quite different from human HBV, especially the duck hepatitis B virus.

However, the biggest disadvantage of the current HBV transgenic mouse model is that the mouse begins to express the viral antigen continuously from the embryonic development stage, resulting in the inability of the mouse to generate an immune response to the viral antigen after birth, that is, the mouse is completely immune to HBV In the state of tolerance, the liver of mice cannot produce immunopathological changes similar to human hepatitis B, which greatly weakens the role of transgenic mouse models in the study of key issues such as the pathogenic mechanism of HBV and the host immune response after HBV infection. Value

Although the Tet-on system has greatly improved compared with the Tet-off system in reducing the toxicity of rtTA and increasing the reaction speed, the problem of the leakage expression (that is, the background expression when not induced) of the system is only higher than that of the Tet-off system. The system has been improved, but the Tet-on system alone is still difficult to overcome the problem of immune tolerance in the embryonic period

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