Cloning, Regulation And Analysis Of Expression On CYP7A1 In Goose

Currently, the research on geese fatty liver mainly focus on the factor such as heredity and environment. However, the molecular mechanism is poorly understood.Synthesis of bile acids from cholesterol plays important role in metabolism of cholesterol. Cholesterol 7 alpha-hydroxylase (CYP7A1) is the rate-limiting enzyme in the synthesis of bile acid from cholesterol.CYP7A1 is a cytochrome P450 heme enzyme, found only in the liver microsomes, it's levels represent the production of bile acids.The gene expression in normal liver and fatty Liver of Landes Geese and Xupu Geese is compared. Genes expressed differently between the two species, fatty liver and normal one were isolated and characterized using mRNA differetial display and fluorescence quantitative PCR. One Expressed Sequence Tag (EST) was found in the process of screening using mRNA differetial display method.By means of rapid amplification of cDNA ends (RACE), the full length cDN A sequences is cloned and compared with GeneBank. The results showed that the homology between that gene and chicken's CYP7A1 gene is 93%. Real-time PCR also showed that the expression of that gene is down-regulated in overfeeding.To further study the effect of CYP7A1 in the occuring and developing of geese fatty liver, the CYP7A1 prokaryotic expression vector pET-28(a)-cyp7al was constructed, transformed into host cell BL21 and induced by IPTG. The fusion protein was purified using Nickel-affinity chromatography column. Mouses were immunized with purified proteins to produce polyclonal anti geese antibody. The purified antibody was used in western blot to study the expression of the gene in fatty and normal geese liver on the protein level. Providing new experimental evidence for understanding the structure and function of CYP7A1 in the forming of geese fatty liver, our research laid the theoretical foundation for the research of lipid metabolism of geese.