Expression Analysis Of Porcine Liver Tissue-specific Gene TTR Promoter-driven BGL1 From Saccharomycopsis Fibuligera In Transgenic Mice

This thesis to verify promoter’s activity and tissue specificity of the swine liver-specific gene TTR level in in living level. In this study, we constructed transgenic mice in which BGL1 gene was expressed driven by the porcine liver tissue-specific gene TTR promoter. We researched that TTR promoter can drive BGL1 expression and the enzyme activity of β-glucosidase by RT-PCR、Western Blot and other technology. The main results are as follows:1. Four founder transgenic mice were generated by pronucleus microinjection technology, three were male mice(A B C) and one was female mouse(D). The founder mice were studied respectively, the establishment line of B founder mouse was successful at last, and obtained a total 5 generations that can be inherited stable exogenous gene, and it was above 90% until F5 generation; exogenous gene copy number was increased form F0 to F3, the copy number reached 112.56 until F3.2. RT-PCR test showed that TTR gene promoter can drive BGL1 gene expression,Western Blot confirmed BGL1 gene can be expressed the corresponding protein in the liver tissue of TG mice, none in other tissue(heart、spleen、lung、kidney、intestines、muscle、fat).3. The enzyme activity of β-Glucosidase in liver tissue of TG and WT were calculated by a β-Glucosidase assay kit, results showed that the relative enzyme activity of TG mouse was significantly higher than WT.4. The liver weight index were calculated respectively in TG and WT, then analyzed for HE staining and EVG staining of liver biopsy、growth curve,results indicated that the expression of exogenous gene did not cause any liver disease in TG mice and have no negative effect on transgenic individual.5. Polyclonal antibody for β-glucosidase of saccharomycopsis fibuligera was prepared successfully, the polyclonal antibody was high titer and has a good specificity by detecting of ELISA and Western Blot.