Proteomics Method Of Hepatitis B Surface Antigen Of Sustained Expression Of The Impact On The Host Cell

The main contributions of this dissertation are:a series of studies focus on the HBsAg Stably expressing cell line have been conducted. The differential proteomic profile of HBsAg stably expressing cell line and its control was analysed. Among the 81 differentially expressed proteins, there were some proteins related to apoptosis regulation; and then autoantibody profile was built up using serological proteomic research and 2DE/MALDI MS. The data provided a new field for the pathogenesis of HBsAg. Glycosylation is a posttranslational modification of many secreted proteins, which was an indication for liver disease. The glycopeptides in the secreted proteins were enriched by a hydrophilic affinity method and then identified by LTQ Orbitrap MS, furthermore, with stable isotope labeling these proteins were quantified. In addition, a new methodology using cationic magnetic nanocomposites for plasma membrane rapid enrichment was constructed in a large scaled proteomic study.Proteomics has burst onto the scientific scene with stunning rapidity over the past few years. By studying global patterns of protein content and activity and how these change during development or in response to disease, proteomics research has boosted our understanding of systems-level cellular behavior and mechanism of disease. In addition, proteomics benefits the identification of new drug targets and the development of new diagnostic markers in clinical research.Hepatitis B virus (HBV), one of the most widely spread viruses, is the prototype of hepadnavirus family. It is estimated that about 350 million people are infected chronically and become carriers of this virus wordwide and 120 million in China. Persistent HBV infection leads to chronic hepatitis, and is closely associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC).In the serum of HBV infected patients,42 nm mature virion particles and 22nm subviral particles can be detected. The later form are composed of the hepatitis B surface antigen (HBsAg) and do not contain viral DNA, usually outnumber the virions by a factor of 1000-fold or more.Serum HBsAg is an important serological marker in chronic hepatitis B. Patients who are positive for HBsAg for at least 6 months are diagnosed as chronic infection. It is difficult to achieve the endpoint of HBsAg clearance. So the pathogenesis and the influence onto the host cells of HBsAg have been long concerned by researchers. The traditional study of pathogenesis and function of HBsAg has focused on individual cell or protein. Proteomics technology allowed us to study global patterns of proteins and activities and how these changes during development or in response to disease, for example the stably expression of HBsAg in our study. HBsAg is a membrane protein that can be secreted by Vesicle system which interferes with normal cellular protein secretion. The study of HBsAg is important but difficult.To solve the above problems, we carried out our research to form this dissertation. The dissertation consists of 5 parts and the contents are summarized as follows:In the first chapter, a review of liver proteomic study and new techniques in proteomics were represented.In the second chapter the HBsAg stably expressing cell line and its control were explored by differential proteomics approach to invest the global effects of persistent expression of HBsAg on host cells. Compared to the control,81 proteins were found differentially expressed in which 38 proteins were up-regulated and 43 down regulated. Some of these results were validated by western blotting. The biological functions of these proteins were analyzed by gene ontology. And some candidate proteins were picked out for further study. We found that replenishing GRP78 improved cellular resistance to apoptosis, whereas reduction of GRP78 by siRNA increased susceptibility even in the absence of HBsAg. Based on these results, more researches will be done.In the third chapter, the autoantibody profile of the HBsAg positive transgenetic mouse by combining serological proteomic approach and 2DE-MALDI MS was constructed for the first time. The results provided a new field for the study of pathogenesis of HBsAg and the discovery of new drug targets.In the fourth chapter, quantitative proteomic analysis of the glycosylation status of the secreted proteins in the HBsAg(+) cell supernatant was performed. Glycosylation is a posttranslational modification of many secreted proteins, and the changes in the glycan structure and amount are an important indication for liver damage. We apply stable isotope labeling by amino acids in cell culture process to quantify the secreted proteins. Then the secreted peptide was enriched by hydrophilic affinity method. The experimental error was minimized by this early labeled step. Many glycosylation changes were detected and it is the first time to exhibit the quantitative profile of the glycopeptides in secreted proteins of the HBsAg stably expressing cell line. It provided early data for further exploration of secreted protein and its glycosylation in the development of chronic liver diseases.In the fifth chapter, a new cationic silica-magnetite nanocomposites based methodology was developed to purify plasma membrane. The plasma membrane provides the physiological and physical boundary between the cells and the surrounding environment, and performs key biological functions such as cell-to-cell recognition and transportation of ions and solutes, as well as acting as receptors for relaying the diverse signals that bombard the cell. The plasma membrane proteins account for about 70% of all known drug targets, while representing 30% of the proteins encoded by the human genome. Consequently, analysis of plasma membrane proteins in various diseased cells is crucial for the discovery of new biomarkers and the design of either therapeutic monoclonal antibodies or small-molecule drugs. The principle of this method, later often adapted and modified, is that the anionic cell surface can bind electrostatically to the cationic colloidal silica. Cationic colloidal silica with a magnetic core (Fe3O4@SiO2-NH4+) was synthesized in our study for plasma membrane proteins enrichment. The enriched plasma membrane proteins were separated by SDS-PAGE, digested in gel and identified by online HPLC-ESI-MS. According to the gene ontology (GO),489 plasma membrane proteins were identified. The purity and the efficiency were distinguished. Notably, among the integral membrane proteins, there were 29 members of the Ras family. Ras is a GTP-binding protein that plays multiple important roles in cell physiological function, including proliferation and inflammatory responses. Given the importance of integral membrane proteins for therapeutic drug design, the ability to enhance analysis of integral plasma membrane proteins with trace samples quickly will facilitate biomarker and therapeutic target discovery.