Research On The Relationship Between Chicken High Mobility Group B1Protein And Avian Leukosis Virus Subgroup J Replication

High mobility group protein B1(HMGB1) was discovered in1973from calf thymus, which is a class of widely present highly conserved non-histone nucleoprotein in eukaryotic cells. HMGB1plays the role of nuclear binding protein under physiological state. When HMGB1is secreated into the intercellular space, it shows the role of late inflammatory cytokine and involved in the inflammatory response. Recent studies have found that HMGB1is not only a kind of transcription and growth factor, but also a kind of important inflammatory cytokines which is related with tumorigenesis, invasion, and metastasis of tumor biological behavior.Avian leukosis disease was caused by avian leukosis virus (ALV), which induced a variety of tumor in the poultry farms. Isolated ALV from chicken can be divided into six subgroups (A-E, J). The subgroup J avian leukosis virus (ALV-J) is one of exogenous avian leukosis virus. ALV-J mainly caused myelocytoma, lymphocyte tumor, hemangioma and other types of cancer in clinical meat type and layers chicken. Futher more, ALV-J infection will cause a rapid decline in laying rate, death and elimination rate rising, and seriously affect the healthy development of the poulty industry.According to the previous proteomic study in our laboratory, chicken high mobility group protein B1(chHMGB1) was the altered host cellular proteins in DF1cells infected with ALV-J. So we speculate chHMGB1may play some roles in the ALV-J infection process. Research on the relationship between chHMGB1and ALV-J repliciation will reveal the role of chHMGB1in ALV-J infection process, which will conducive to a comprehensive understanding of the mechanism of ALV-J infection. And it could provide a theoretical basis for the treatment and prevention of ALV-J also. 1. Establishment and application of the SYBR Green I Real-time PCR assay for detection of chicken high mobility group B1geneIn order to detect the change of chHMGB1gene expression level during the process of ALV-J infection, we designed specific chHMGB1primer pairs for the Real-time PCR assay according to the sequence of chicken HMGB1. With the primer pair, we amplified chHMGB1gene fragment and ligated target gene with pGEM-T vector to make the standard chHMGB1gene target. After gradient dilution the plasmid vector of chHMGB1gene and amplification chHMGB1gene standard curve by Real-time PCR, the absolute quantitative Real-time PCR assay for identify chHMGB1was established. And the chHMGB1gene expression level in the different tissues of one day-old chicken and the normal cell lines from avian was caculated by the Real-time PCR. The results provided a rapid and reliable detection method, which will useful for the further biofunctional study of chicken high mobility group B1in immunity and inflammation process of poultry dieases.2. ChHMGBl and virus gene dynamic changes in ALV-J infected cells and tissuesBy Real-time PCR, indirect immunofluorescence and Western Blot experiments, we detected the gene and protein dynamic expression levels of chHMGB1and virus gene in the CEF, DF1and HD11cells infected with ALV-J.Real-time PCR results showed that with increasing time of infection, ALV-J replication in different avian cells raised significantly. In the process of virus infection, the chHMGB1gene in DF1cells showed upregulation at1h, downregulation at6h, upregulation at12h,24h,48h and72h was downregulated, and rising again at96h time point. The chHMGB1gene in CEF cells was upregulated at1h and6h, and downregulated at12h. The expression level at24h,48h,72hand96h were upregulated; The chHMGB1gene in HD11cell was downregulated at1h and6h, upregulated at12h, downregulated at24h and48h,and then upregulated at72h and96h.Indirect immunofluorescence results showed that with increasing time of infection, virus gene fluorescence increased gradually. But in different cells, the chHMGB1protein was stable in nucleus, without the occurrence of transfer. The results of western blot were basically consistent with Real-time PCR test results.The results of the kidney and liver tissues of the SPF chicken infected with ALV-J at1,28,56,84,112and140days revealed that in the kidney chHMGB1and viral gene had almost the same change trend. And chHMGB1genes expresssion in liver tissue reached a peak at28days, and then fell rapidly and maintained the normal level. The virus gene expression had been in a state of a wave in liver tissure.In the ALV-J infected chicken peripheral blood mononuclear cell parten, Real-time PCR results showed that the trends of the chHMGB1and virus gene were exactly the same with different strains of ALV-J, which were increased first and then decreased.3. Effect of chHMGBl on subgroup J avian leukosis virus replication in cellsTo investigate the effect of chHMGB1on viral replication, function inhibitors of HMGB1glycyrrhizin (Glycyrrhizin, GR) was used. In this study, the infection ALV-J experimental groups were divided into adding GR group and without the GR group. By Real-time PCR and indirect immunofluorescence assay, the results demonstrated that ALV-J virus replication cells increased significantly in DF1and CEF after treatment with GR. So the chHMGB1in DF1and CEF cells might block virus replication. But we found the opposite results in HD11cells. These results provided a reference for further study of chHMGB1roles in the ALV-J disease development.