Preliminary Study On The Biofunction Of Casid Protein P27 Of Avian Leukosis Virus

Avian Ieukosis disease was caused by avian leukosis virus (ALV), which induced a spectrum of different neoplasms and severe immunosuppression also. In addition to direct damage caused by a viral infection in chickens, avian leukosis disease cause decreased productivity, mixed infections, secondary infection, huge economic losses and serious harm to the development of the poultry industry. The p27 gene coded by Avian leukosis virus is a highly conserved gene among all the different ALV subgroups. Viral capsid protein encoded by p27 gene accounts for more than 30% of the total protein component of the virus. It is the group-specific antigen (GSA) of Avian leukosis virus with many easily detected antigenic site in it. These information indicate that p27 protein may play some role in the pathogenisis of ALV. In current study, we established an absolute quantification Real-time PCR assay for detection of Avian Leukosis Virus p27 gene, which provides a good technology for the further bio-functional study of ALV-p27. And then, we studied the capsid protein on the cell immunosuppression by transient transfaction the eukaryotic expression vector of Avian Leukosis Virus p27 into the cells. At last, we tried to idetified the interacted protein in the host cells by co-IP and silver stain. All the studies provide clues and theoretical basis for further study of the molecular mechanism of ALV-p27 protein and host interactions.1. Establishment and application of the SYBR Green Real-time PCR assay for detection of Avian Leukosis Virus p27 geneFirstly, we designed specific p27 primer pairs for the Real-time PCR assay according to the sequence of chicken p27 gene. With the primer pair, we amplified p27 gene fragment and ligated target gene with pGEM-T vector to make the standard p27 gene target. After gradient dilution the plasmid vector of p27 gene and amplification p27 gene standard curve by Real-time PCR, the absolute quantitative Real-time PCR assay for identify p27 was established. And the ALV-p27 gene distribution and copies in the different tissues of one day-old chicken which were injected by virus at the 6 ED embryos were characterized by the real-time PCR assay. The results provided a rapid and precise quantitative detection method for the virus replication, and a useful technology supplementation for the ELISA method of ALV-p27 detection. The developed absolute quantification Real-time PCR assay provides a good technology for the further bio-functional study of ALV-p27.2. ALV-p27 protein suppress cytokines expression in host cellsIn order to study the effect of ALV-p27 protein to the cellular immune response of DFl which were stimulated by Lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (polyl:C) after transient transfaction with ALV-p27, we inserted the p27 gene into the pcDNA3.1(+) vector to make the eukaryotic expression vector of pcDNA3.1-p27. After transient transfaction with pcDNA3.1-p27 in DFl cells, we stimulated the cells with LPS and poly(1:C) respectively and harvested the cells after stimulation for 1,6,12,24 and 48 hours. Then, the real-time PCR assay were carried out to detect the inflammatory cytokines and anti-viral cytokines genes expression level. The results demonstrated that the p27 protein expression significantly inhibited the gene expression of IFN-β,IL-6, IL-12 and other cytokines compared with normal vecetor transfection group. And the gene expression of key molecules in the signaling pathway, MYD88 and IRF7, were inhibited by p27 protein also.All the results revealed that the ALV-p27 might induce immunosuppression through inhibition the expression of inflammatory cytokines and anti-viral cytokines genes. But the detail molecular mechanism need to be addressed in the further study.3. preliminary Screening the proteins interacting with ALV-p27 protein in host cell.In order to screen the proteins which interact with ALV-p27 protein in susceptible cells, the monoclonal antibody 5D3 specific to ALV-p27 was used to co-immunoprecipitated in DFl cells transient transfected with pcDNA3.1-p27, HD11 cells and DF1 cells infected with ALV-J respectively. The different candidate protein bands were subjected to Mass spectrometry (MS). Two distinct bands which molecular weight were about 71kd and 72 kd were precipitated from among all the three co-immunoprecipitation assay. The two proteins were idetified as 72kd glucose-regulated protein (GRP78) and Heat shock cognate 71 kDa protein (HSPA8). In addition, another two distinct bands which molecular weight were about 73 kd and 60 kd were precipitated from two co-immunoprecipitation assay, which were idetified as 73kd Heat shock cognate protein (HSPA9) and ATP synthase subunit alpha 60 kDa protein (ATP5A1). After comprehensive analysis of all MS, the results suggested that the four candidate proteins GRP78, HSPA8, HSPA9 and ATP5A1 might interact with ALV-p27 protein in host cell and play a certain role in the bio-function of ALV-p27 protein. All the results in this section provide a credible candidate proteins and important theoretical basis for further study of ALV-p27 protein bio-functions and features.