Identification Of The Dominant Protein And Its Interaction Proteins Of VSV That Induces Apoptosis In N2a Cells

Vesicular stomatitis virus(VSV) is a member of the Vesiculovirus Rhabdoviridae, mainly causes vesicular stomatitis of pigs, horses, crows and some wild animals. The characteristic vesicles are observed on the tongue, lips, buccal mucosa, nipple and coronet. Vesicles could be broken easily, exposing the red erosion granulation tissue. Human could be affected occasionally by direct contact with VSV, showing the flu-like and other symptoms such as fever, sensation of chill, headache, nausea, courbature and inability. The disease has been listed as legal communicable disease by Office International Des Epizooties. In addition to serious vesicular stomatitis, its damage to the nervous system gradually attention by the researchers. Like other neurotropic virus, vesicular stomatitis virus can spread along the axon anterograde and retrograde, mainly cause central nerve damage. Through the brain vaccination can cause mouse brain nerve cell necrosis and mononuclear cell infiltration quickly. The damage of mouse nerve cell is closely related to the apoptosis. M protein is one of the structure protein of VSV, which as an important virulence factor plays an important role in the process of VSV induced disease development. To the other susceptible cell research found that M protein not only plays an important role in the process of inducing the accumulation of VSV and causes cytopathogenic effect of cells infected with VSV but also participates in the apoptosis process induced by VSV and antiviral immunity of host cells. However, the role of neurotropism of VSV in the process of VSV infection is unknown. To this end, this thesis has carried out the research work.VSV can replication on the N2 a cell and induces obvious CPE on the infected cells. So in the study, N2 a cells was selected as viral infection model. Firstly, the apoptosis of N2 a cells induced by VSV were detected by the observation of morphology, PCR identification, dyeing by Hochest, DNA Ladder and flow cytometry test. The results showed that VSV could induce the apoptosis of N2 a cell. Then, we constructed the eukaryotic expression vector of matrix protein, glycoprotein protein and phosphoprotein. The apoptosis of N2 a cells were detected after transfection with the eukaryotic expression plasmids as mentioned above. The results showed that the cell apoptosis induced by matrix protein higher than glycoprotein protein and phosphoprotein. It can be seen that VSV-M protein is one of the leading proteins to induce apoptosis of N2 a cells.The bait expression vector was constructed using M protein as a bait. The bait expression vector was transformed into AH109 yeast cell by yeast two hybrid technology in order to test the toxicity and the activation. The detection results showed that further study could be performed. We performed the hybrid fusion using the bait vector and the library of mouse brain. Positive clones were obtained by preliminary screening using the auxotroph medium. The plasmids extracted from positive clones were transformed into E.coli. The plasmids were further identified by PCR and sequencing. The interaction proteins such as carboxypeptidase E and HAX-1 were determined by comparing with NCBI database.HAX-1(HS1-associated protein X-1) could interact with hematopoietic cell specific Lyn substrate1. The molecular weight of HAX-1 is 35 k Da, which could interact with many proteins. HAX-1 may participate in other different process including the transduction of B cell signal, receptor-medidated apoptosis, proliferative response, and so on. In recent years, HAX-1 is involved in more and more research, however, there is not associated with VSV infection. In order to verify the interaction between HAX-1protein and VSV-M protein, we performed the test of rotary hybrid. The HAX-1 gene was amplified by PCR, and the corresponding eukaryotic expression vector was constructed. The HAX-1 protein was expressed and used the preparation of HAX-1 polyclonal antibody. Then, we further confirmed the interaction of HAX-1 protein and VSV-M protein using GST pull-down and co-immunoprecipitation. The colocalization of HAX-1 protein and VSV-M protein was observed by laser confocal microscope. The study on the interaction between VSV-M and HAX-1 would not only establish foundation for exploring the new function of M protein at the molecular level, but also help to further clarify the pathogenic mechanism of VSV.In summary, this study confirmed that VSV can induce neuronal apoptosis, and for further analysis of the molecular mechanism of VSV induced neuronal damage added more experimental data.