Identification Of Cellular Binding Protein Of Hepatitis B Virus PreS1 Domain On HepG2 Cell Membrane

ObjectiveTo identification of cellular binding proteins on HepG2 cell membrane and lay the foundation for further exploration and study of cellular specific receptors for Hepatitis B virus (HBV) envelope protein and invasion mechanism of HBV through study of separation of HepG2 membranin bound to preS1 followed by MS identification, and of interaction between the identified protein and preS1 in vitro and in vivo.Methods1. Separate preS1 binding protein on HepG2 membrane: preS1 gene was recombined with GST tag and expressed in prokaryotic cells; the fusion protein GST-preS1 was incubated with precleared lysate of biotinylated HepG2 cells and GST pull down assay was employed to detection of preS1 domain cellular binding protein.2. Identify the separated preS1 binding protein by MS methods: MS identification was performed in Ion Trap LC/MS system and protein identification was performed in Spectrum MILL for MassHunter Workstation against the Swiss-Port protein database search engine with species limitation of Homo sapiens.3. Express the identified protein in prokaryotic cells: gene of identified protein obtained from database was recombined with His tag and expressed in prokaryotic cells using gene recombination technology.4. Observe the distribution of the identified protein in HepG2 cells using laser scanning confocal microscope (LSCM) and verify the interaction between preS1 and the identified protein at cell membrane level by FRET technique.5. Further confirm the reciprocal action of preS1 and the identified protein by in vitro test.Results1. Recombinant expression plasmid pGST-preS1 was constructed and expressed in prokaryotic cells successfully. GST-preS1 fusion protein was purified using affinity chromatography methods with purity of 90%. Through acting with its specific antibody, GST-preS1 showed good antigenicity.2. An approximately 110 kDa protein (p110) on human hepatoma cell line HepG2 plasma membrane was shown to bind to the preS1 domain of the fusion proteins by pull down test.3. The 110kDa band was cut off from SDS-PAGE gel and subjected to in-gel digestion with Trypsin followed by LC/MS/MS analyses performed in an Ion Trap LC/MS system. Two proteins which is 78 kDa glucose-regulated protein precursor (GRP78) and LanC-like protein 1 (LANCL1) were identified.4. Recombinant expression plasmids pW28-GRP78 and pW28-LANCL1 were constructed and expressed in prokaryotic cells successfully. His-GRP78 and His-LANCL1 fusion protein was purified respectively using affinity chromatography methods with purify of 95%. Through acting with their specific antibody, His-GRP78 and His-LANCL1 showed good antigenicity.5. The uniform distribution of GRP78 on HepG2 cell membrane under LSCM demonstrated that it is one of the HepG2 cell membranin.6. Colocalization of GRP78 and preS1 on HepG2 cell membrane was observed under LSCM after preS1 incubated with HepG2 cell and the interaction between each other was verified by FRET technique.7. In vitro test further confirmed the interaction between GRP78 and preS1.ConclusionGRP78 is one of the membranin of HepG2 cell interact with HBV preS1 domain.