Preliminary Identification Of Bile Salt Export Pump(BSEP) In Snake Liver

Objective: Snake bile is a normal Traditional Chinese Medicine. This research focuses on the principal transporter in charge of bile acid secretion- bile acid export pump(BSEP) in snake liver to identify BSEP in genetic and protein level. That could built up foundation for further study on bile metabolism and regulation.Methods: In genetic level, specific primers were designed based on bioinformatic prediction on genes coding BSEP in three kinds of snakes(Ophiophagus Hannah, Thamnophis sirtalis and Python bivittatus), PCR technique together with homology comparison was used to identify BSEP in snake liver; based on actual sequence detected, 3′RACE and 5′RACE(rapid-amplification of c DNA ends) were applied to obtain whole sequence of ABCB11 from Elaphe carinata; in protein level, nano-RPLC was used to fractionate proteins extracted from snake liver into 15 fractions, followed by triple-TOF analysis on ion information and search against Ophiophagus Hannah database to be further analyzed by proteomics.Results: 4 sequence in ABCB11 were obtained through Polymerase Chain Reaction combined with homological analysis. Employing the technique of 3′RACE, unkown sequence close to 3’-end was identified and spliced together with 4 former sequence(2666bp), the splicing sequence has high homology compared with sequences predicted in three other kinds of snakes; no reasonable results were acquired from 5′RACE experiments, improvements need to be done with it. RPLC-MS/MS results indicate that 11 peptides in BSEP have confidence greater than 95%, protein ranks 336 across the protein list, false discovery rate(FDR) is 1%, the key parameter-unused protscore is 22.56, indicating an accurate existence of BSEP.Conclusion: The existence of BSEP has been successfully validated from genetic and protein level. This result build foundation for research of protein structure and function, as well as regulation for bile acid in snake.