The Prokaryotic/Eukaryotic Expression And The Functional Research About The Hepatocyte Membrance Receptor Protein GRP78

The glucose-regulated protein precursor 78 (GRP78), also referred to as BiP (immunoglobulin heavy-chain binding protein), was firstly found from the endoplasmic reticulum. GRP78 is involved in major biological functions of the regulation of calcium balance, newly synthesized protein in the endoplasmic reticulum, protein transmembrane migration, folding, maturation, transport and reverse transport, etc. In addition to the endoplasmic reticulum, GRP78 was also expressed in the cell membrance. Some researchers proposed GRP78 on the cell membrance could also be existed as a receptor and signal transduction molecules. Later research had confirmed the view: GRP78 was the hepatocyte membrane of the type 2 dengue virus receptor complex components, was also co-A9 Kosac virus receptor molecules. All these studies had showed that GRP78 on the cell surface had signal transduction and antigen-presenting function.Hepatitis B virus (HBV) infection is a serious threat to human life and the global public health problem. Despite considerable advances in the understanding of natural history of HBV disease, the process of its replication and antigenic structure, most of the early steps in the virus life cycle remain unclear for lack of an in vitro infection system. HBV attatching to permissive cells, fusing and penetrating through cell membranes and releasing subsequent genome are largely a mystery. Current knowledge on the early steps of HBV infection shows that HBV binding to the special recptors on human hepatocyte membrane via preS1 domain of large surface protein (LHBs) and then fusing with cell membrane trigger the viral infection. Our discussion group in the preliminary studied with recombinant protein GST-preS1 fusion protein for the probe, using pull-down method to direct isolate the HepG2 membrane protein binding to preS1. Further analysis by mass spectrometry identified GRP78 was the binding protein.Observed the distribution of the GRP78 in HepG2 cells using laser scanning confocal microscope (LSCM), we found that in normal conditions, GRP78 was expressed both on cell membrane and cytoplasm in HepG2 cells. The full-length cDNA of GRP78 was obtained from HepG2 cells by RT-PCR. Express GRP78 and its section expressing proteins in prokaryotic cells: gene of GRP78 obtained from database was recombined with His tag and expressed in prokaryotic cells using gene recombination technology. Recombinant expression plasmid PW28-GRP78, PW28-GRP78N, PW28- GRP78C was constructed and expressed in prokaryotic cells successfully. These fusion proteins were purified using affinity chromatography method with purity of 90%. The fusion protein GST-preS1 was incubated with GRP78 and its section express proteins. Pull down assay and consensual ELISA were employed to detection the interaction between preS1 and GRP78. The results showed that preS1 combinated with GRP78, especially GRP78N section, from the impact with His tag, and with the HBV virus in a dose-dependent. Recombinant plasmid pcDNA3.1-GRP78 was proved to be successfully constructed by restriction enzyme digestion analysis and DNA sequencing. Western blotting showed that the protein level of GRP78 in pcDNA3.1-GRP78 transfecion group was higher than that in untransfection group and pcDNA3.1 transfecion group. The data proved that GRP78 was overexpressed in pcDNA3.1-GRP78-transfected HEK293 cells in protein level.Taken together the results suggest that GRP78 may be a crucial molecule in the viral entry machinery.