The Study Of Panning And Validation Of Protein Target About Hepatitis Treatment By Oxymatrine

ObjectiveThe main active ingredient Oxymatrine of herbal Sophora have the treatment effect for hepatitis B.Some protein molecules was involved in the mechanism of Oxymatrine.It is still not clear whether these molecules are a direct target or subsequent reaction of oxymatrine effect.In order to find the.target of Oxymatrine,this study aims to reveal the direct binding protein of Oxymatrine target by Molecular biology and bioinformatics under the guidance of TCM theory.MethodsA T7 phage cDNA library of chronic hepatitis B of liver and gallbladder damp-heat syndrome was build and biopanned by Oxymatrine for binding protein targets.UQCRB(ubiquinol-cytochrome c reductase binding protein)was selected for further study.The UQCRB full-length gene was cloned into PET-28a(+),and was expressed and purified.The interaction between UQCRB and Oxymatrine was validated by affinity between oxymatrine binding phage belonging to UQCRB gene sequences and molecular docking and ITC titration of UQCRB and Oxymatrine.ResultsThe initial titer of T7 phage cDNA library of chronic hepatitis B of liver and gallbladderd damp-heat syndrome is 3×105pfu/ml and amplified library titer is 1.8×109pfu/ml.After four biopanning Oxymatrine positive group compared Oxymatrine negative group titers showed enrichment.Sequence analysis of Oxymatrine binding phage by biopanning belonged to 12 kinds of protein genes,and 4 clons was unidentified.25 clones belongs UQCRB protein gene,and the number of clones was at the top.Prokaryotic expression vector of UQCRB full-length gene were constructed,expressed and purified.And the results were confirmed by restriction enzyme digestion and agarose gel electrophoresis,SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue R-250 staining.The affinity of oxymatrine binding phage belonging UQCRB gene sequences and oxymatrine was measured.The number of phage binding of each group was analyzed by minitab statistical analysis software.one-way ANOVA F=6.61,P =0.015,far less than 0.05,TBST group,biotin group,oxymatrine and matrine group group difference was statistically significant,Tukey mean(standard deviation based on the merger)with 95%confidence interval single group and Tukey 95%confidence interval for Treatment and all paired comparison oxymatrine group with TBST group,biotin group and matrine group difference was statistically significant.By Oxymatrine UQCRB computer molecular modeling and docking we obtained a possible conformation which suggested the interaction between UQCRB and oxymatrine.Its binding energy was of-6.27 kcal/mol,involving receptor residues Tyr21,Arg33,Tyr83,Glu84,Asp86,Pro88,Glu91.There was a hydrogen bond between 1-0 radical of the oxymatrine and OH of TYR-83 residue.ITC titration results showed that there were some certain thermodynamic effects between UQCRB and Oxymatrine.ConclusionT7 phage cDNA library of chronic hepatitis B of liver and gallbladderd damp-heat syndrome was successfully established.And polypeptides library which may be combined with Oxymatrine was obtained.After the construction of prokaryotic expression vector of UQCRB,its prokaryotic expression and purification was conducted.Combination of UQCRB and Oxymatrine was confirmed by affinity and identification,molecular docking methods and ITC UQCRB is likely to be the target of oxymatrine.