Identified The Key Areas In Form Aggresome Of PRRSV Nsp2

Porcine reproductive and respiratory syndrome(porcine reproductive and respiratory syndrome, PRRS) is composed of porcine reproductive and respiratory syndrome virus(porcine reproductive and respiratory syndrome virus comes, PRRSV) immunosuppression caused by disease, the main cause of pregnant sows miscarriage, premature birth and dead fetus, such as comprehensive reproductive disorders, piglet and fatten pigs developed respiratory syndrome, pathogenic mechanism is not clear because now the disease and no effective drug or vaccine application, progressively the outbreak of the disease and cause huge economic losses to the global pig industry.Aggresome as a gathering place and viral proteins form, provide rich material basis for viral replication and assembly, and can avoid the identification of the immune system without degradation, cell protein is one of the important proteins involved in aggresome form 14-3-3. PRRSV nonstructural protein Nsp2 encoded by ORF1 a domain with complex structure and function of the protein. The basis of previous studies, found that after Nsp2 protein in vitro expression can occur together form aggresome. In order to further study the structure of nsp2 expression and aggregation formation area, will be truncated nsp2 protein expression analysis, looking for key areas related to the aggregate formation, provide theoretical basis for the design of the vaccine.According to the structural protein nsp2 protein, it is divided into OTU, HV, TM and Tail 4 segments. Cloning respectively with green fluorescent protein(GFP) label of eukaryotic expression vector, the sequence determination and Western blot for identification of protein expression. Will build the recombinant plasmid transfection of PRRSV susceptible Marc- 145 cells, not susceptible to BHK cells- 21 cells, expressed in confocal microscopy of nsp2 protein formation aggresome situation, found that the formation of aggresome are associated with high variable area and carboxyl terminal tail. Previous studies also found that nsp2 and the protein interaction with 14-3-3, 14-3-3 proteins play an important role in the process of aggresome formation. With 14-3-3 different subtypes of antibody were positioning analysis found that nsp2 with alpha/beta and epsilon subtypes of protein interaction with 14-3-3. For further research on aggresome formation mechanism, the truncation of nsp2 protein transfection 293 t cells and Marc- 145 cells, via the pull- down and Western blot analysis found that the 14-3-3 and the change of nsp2 high interaction, further confocal microscope found that alpha/beta and epsilon subtypes of 14-3-3 proteins and nsp2 high variable area and carboxyl terminal tail has the effect of positioning. With the alpha/beta 14-3-3 subtypes of antibody in immune coprecipitation, precipitation product on simple analysis of detected nsp2 protein.The above results show that nsp2 expression form of aggresome with alpha/beta and epsilon subtype of the 14-3-3 proteins, nsp2 high variable area and carboxyl terminal tail about aggresome formation, these two areas form micelles with alpha/beta, epsilon subtypes of the 14-3-3 proteins. The discovery of these results lay a foundation for further study of PRRSV pathogenesis mechanism, at the same time as the research provides the theory basis for prevention and control of PRRSV drug and vaccine design.