Construction Of An Animal Model For The Regulation Of HBV Gene In Lentivirus System

Hepatitis B is one of worldwide epidemicdiseases endangering human health. It was caused by Hepatitis B virus, At present, Approximately 350 million people infected with hepatitis B virus worldwide. HBV infection has revealed clinical coursesfrom asymptomatic infection to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC).China is a high prevalence area of hepatitis B virus. Hepatitis B virus belongs to the hepatotropic DNA virus, only infected in primates, After being developed for years, Hepatitis B still can not be cured. So Study on the cytological and molecular mechanism of HBV specific immune tolerance is the right important research direction.Study on the cytological and molecular mechanism of HBV specific immune tolerance needs to break the bottleneck caused by animal model. However, current animal which can used to study HBV are unsuitable to construct animal model due to itself limitation, such as chimpanzee, mouse, groundhog and duck etc. Importantly, these models do not reflect dynamic process of intracellular change during HBV infection and unavailable to study the difference between before and after the formation of immune tolerance. This is a result of the static compatibility between viruses and cells because DNA of HBV had integrated in genome of hepatocyte. The key to break this tolerance is to find out the difference of immune system, such as specific molecular and diversity of target. So we used Tetracycline-controlled lentiviral system to construct transgenic mice, can control the expression of HBV genes on different time and site. Based on this model, the cytological and molecular mechanism of HBV specific immune tolerance could be researched on various periods of development and immune state.In previous studies, The tetracycline regulating expression plasmid containing seven HBV genes (X,P, C, preC-C, S, preS2-S, preSl-S2-S) were constructed. The plasmids weretransfected into the lentiviral packaging cell line HEK293T cells, and the virusparticles containing the regulating gene pLVX-Tet-On and the expression gene pLVX-LS(X) were gained. The virus titer was about 108LPs/ml. After injection 10 ul virus mixture in each mice testis, mice were caged together with female mouse. When the F0 generation mice were born, their genome was extracted. The degree of genome integrality was identified by southern-blot and inverse PCR. We choose the mice, containing both Tetracycline-controlled gene and HBV-X gene, to induce the protein expression. After 72 h induction, total RNA were extracted for RT-PCR, identify the expression of these genes. Then, we used western-blot and immunohistochemistry assay to verify the protein expression of these genes in mice live.Result:A total of 96 mice received in this study. We analyzed the mice genome by Southern-blot and Inverse PCR, Regulator gene Positive mice only 14, positive rate is 14.58%.HBV-X gene Positive mice only 5, and positive rate is 10.20%. Then inducible positive mice transgene expression; Add 1.5mg/ml Dox in the water of mice for 72h in order to inducible transgene expression. After 72h, killed the mice and extract protein from the mice liver. We analyzed the mice protein by western-blot and immunohistochemistry. Finally we found HBV-X protein expressed in mice organization.