The Preliminary Study Of Interaction Between HBV Large Protein And Homo Sapiens Pancreatic CDK5RAP3 Protein

Objective:To further study the interaction between HBV large protein and the host pancreatic cell.Methods:1.We transformed yeast cell expression vector pGBKT7-LHBs into yeast strain AH109 and performed yeast two hybrid by mating AH109 containing LHBs gene with Y187 containing plasmids of human pancreas cDNA library and screened of proteins binding to HBV large protein from human pancreas cDNA library.2.Homo sapiens CDK5RAP3 gene was amplified with reverse transcription polymerase chain reaction(RT-PCR). 3.We constructed eukaryotic expression vector pcDNA3.1-myc-his- CDK5RAP3 and confirmed the interaction between HBV large protein and Homo sapiens CDK5RAP3 protein using co-immunoprecipitation and Western blotting.4.We constructed eukaryotic expression vector pACT-CDK5RAP3 and confirmed the interaction between HBV large protein and Homo sapiens CDK5RAP3 protein using mammalian two-hybrid experiment.5. We constructed eukaryotic expression vector pDsRed1-N1-CDK5RAP3 and transfected into Capan2 cell lines by using lipofectamine,then observe the location by confocal fluorescence microscope. Results:1.We found eleven proteins which interact with HBV large protein,including Homo sapiens pancreatic lipase,Homo sapiens carboxypeptidase B1,Homo sapiens protease, serine, 2 (PRSS2),Homo sapiens histocompatibility (minor) 13 (HM13),Homo sapiens aconitase1,Homo sapiens elastase 2A (ELA2A),Homo sapiens ribosomal protein L10a (RPL10A)and so on.2. Homo sapiens pancreatic CDK5RAP3 gene was obtained .3.We constructed eukaryotic expression vector pcDNA3.1-myc-his- CDK5RAP3,which was cotransfected into Capan 2 cell line with pcDNA3.1(-)LHBs and confirmed the intra-cellular interaction between HBV large protein and homo sapiens colipase protein with Co-immunopercipitation.4.We constructed eukaryotic expression vector pACT-CDK5RAP3,which was cotransfected into Capan 2 cell line with pBIND- LHBs and confirmed the intra-cellular interaction between HBV large protein and homo sapiens CDK5RAP3 protein with mammalian two-hybrid.5.To investigate the subcellular localization of LHBs and CDK5RAP3, the plasmids expressing LHBs and CDK5RAP3 fusion protein with pEGFP-C1 and pDsRed1-N1 were constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion. After 48 hours, the expression was detected by confocal fluorescence microscope. Conclusion:We found eleven proteins which could bind to HBV large protein and confirmed the interaction between HBV large protein and homo sapiens CDK5RAP3 protein in intra-cellular , which laid the foundation for further studying the mechanism of HBV large protein.