The Panning Of Proteins Target Binding To Matrine And Cloning Expression And Purification Of Apoc?

ObjectivePhage display technology can be used for seeking drug targets,Which has been reported.In this paper,in order to explorie the mechanism of matrine,we tried to biopan the polypeptide targets to which matrine direct binds by using phage display technology and T7 phage cDNA library of chronic hepatitis B,and verify the full length of targets,so that can lay foundations for further researches on mechanism of matrine in treatment of hepatitis B.MethodsPhages in T7 phage cDNA library of chronic hepatitis B were selected by repetitious biopanning?affinity ? elution ? amplification ? affinity?in 96-well plates of avidin,with biotinylated matrine as the target molecule.The selected phages were then verified,sequenced and peptide analyzed.The sequence was then blast aligned in GenBank,and also analyzed the possible binding peptides.Apolipoprotein C? was chosen to do further research for its abundant cloning,whose genes will be cloned,expressed and purified.Later,cloning vector of PMD^TM19-T-ApoC? was constructed and validated by gene sequencing.Subsequently,a plasmid vector of PET28a carrying target genes?PET28a-ApoC ??was constructed and transformed into BL21?DE3?Star and fusion expressed.The expressed products were purified by Ni NTA resin affinity chromatography and later verified by western-blotting.ResultsAfter four rounds of screening,phages were gradually enriched and remained relatively stable.The T7 phage was randomly selected and sequenced to get the sequence of matrine binding polypeptide and protein,which also verified by reverse affinity.Cloning vector of PMD^TM19-T-ApoC I and expression vector of PET28a-ApoC? were both successfully constructed in our experiments.Recombinant plasmids were constructed,sequenced exactly and introduced into BL21?DE3?Star.After optimizing conditions,the target genes carried by plasmids can be induced by IPTG to express in BL21?DE3?Star,and the purity is verified by western-blotting.ConclusionsT7 phage display technology is a high-throughput selection technique which can effectively biopan the ligand specifically binding to target molecules.In our experiments,we have biopanned the phaseolin polypeptide which binds to matrine,and successfully expressed and purified ApoC-?,thus providing new ideas and foundations for further studies on mechanism of matrine in treatment of hepatitis B.