| Beta-catenin (β-catenin) protein is a kind of multifunctional proteins that can regulate cell adhesion and act as a key molecular in Wnt/β-caten in signaling pathway. The β-catenin shows different roles when it locates in different parts of the cell. On the surface membranes of cells, β-catenin is combined with E-cadherin to form solid adhesion between cells and maintain constructive in tissues and cells. In the cytoplasm, one part of the β-catenin has good adhesion to E-cadherin, the rest of β-catenin combines with APC, GSK3β and Axin to form a protein complex, which phosphorylates β-catenin protein to degrad in the cytoplasma.When the Wnt/β-catenin signaling path way is activated, β-catenin will migrate into nucleus and conbine with TCF/LEF to promote downstream gene, such as c-myc and cyclin Dl, duplication, and promote the malignant transformation of cells. The activation of the Wnt/β-catenin pathway affects cell growth, proliferation, apoptosis and so on. More and more studies show that abnormal expression and nuclear transfer of β-catenin is associated with a variety of human tumor diseases, such as liver cancer, colon cancer, breast cancer and so on. But the association between avian oncogenesis diseases, such as avian leukosis and marek’s disease, and the biological function of β-catenin is remain to be clarified.In current study, we generated anti-avian β-catenin affinity purified polyclonal antibody and established an absolute quantification real-time PCR assay for detecting avian β-catenin with β-catenin standard curve. We also observed β-catenin localization after CEF and DF1 cells treated with GSK3β inhibitor and LiCl respectively. At last, we detected gene expression level of the target and key genes in the Wnt/β-catenin signaling pathway. All the study provide a theoretical basis for further research on the relationship between avian oncogenesis diseases and Wnt/β-catenin signaling pathway.1. Generation and identification of polyclonal antibody against chicken beta-cateninTo prepare and identify the polyclonal antibody against chicken beta-catenin(β-catenin), the specific peptides was selected by comparison and analyzed according to the sequence of β-catenin published in GenBank, conjugated with keyhole limpet hemocyanin (KLH) before immuizing New Zealand rabbits. The immuized rabbit serum was purified by protein A column. Then, the specificity of the affinity-purified polyclonal antibody was identified by Western blot, IFA and immunohistochemistry assay. The polyclonal antibody harvested from New Zealand rabbits was demonstrated being specific to detect the natural β-catenin protein in avian cells, and could be efficient in Western blot, IFA and immunohistochemistry assay. The results indicated that the affinity-purified polyclonal antibody which could bind chicken β-catenin protein had been successfully generated.2. Establishment of an absolute quantification real-time PCR assay for detecting avian beta-catenin geneA pair of PCR primer was disigned according to the published sequence of β-catenin in GenBank. The desired fragment was amplified from cDNA of CEF. After β-catenin fragment was inserted into pGEM-T-vector and sequence analysis, then the fragment was subcloned into pcDNA3.1 expression vector. The positive recombinant plasmid was screened and confirmed by double enzyme digestion. The results demonstrated that the pcDNA3.1-β-catenin recombinant construct was successfully made, which was used as standsrd plasmid in the further real-time PCR assay. An absolute quantification real-time PCR assay for detecting avian P-catenin gene was established by using the designed specific β-catenin primers for real-time PCR and the recombinant construct as standard plasmid DNA. The β-catenin gene copies in different tissues of one-day old SPF chicken were characterized by real-time PCR assay. The results indicated that among the different tissues of one-day old SPF chicken, copy number of β-catenin gene in brain was the highest, the lowest number was in the bursa of fabricius. The established absolute quantification real-time PCR assay could provide an aviable method for the further bio-functional study of P-catenin.3. The location of beta-catenin in poultry cellsAccording to the published researsh, the β-catenin in mammalian cells is degradated in cytoplasm of cells in normal conditions. And it will migrate into the nucleus when it can not be degradated and gather in the cytoplasm. To investigate whether β-catenin in poultry cells has the same feature, we transfected CEF cells with recombinant β-catenin eukaryotic vector, treated cells with GSK3β inhibitor and LiCl respectively to observe the expression and localization of P-catenin in cells by IFA. At the same time, the key genes if the Wnt/p-catenin signaling pathway were detected by the designed primer pairs also. The results showed that β-catenin transfer into nucleus after transfection for 24 hours and treatment by GSK3P inhibitor and LiCl respectively. The high expression level of p-catenin gene in DF1 cells were LEF and WIF genes, in CEF cells were also observed. All the results demonstrated that β-catenin in avian cells has the same feature as it in mammalian cells, which would transfer into the nucleus when it gathered in cytoplasm to active transcription gene.4. Expression of Wnt/β-catenin signaling pathway genes in chicken tissues and the location detecting of P-cateninFor the study of the association between Wnt/β-caten in signaling pathway and avian oncogenesis diseases, RNA of virus infected chicken tissues and clinical chicken tumor tissues samples were extracted and reverse transcripted to cDNA. The SPF chicken tissues with the same age were used as normal control. The real-time PCR assay was carried out to detect the gene expression level of Wnt/p-catenin signaling pathway. The results showed that in kidney samples of ALV-J infected group the β-catenin gene expressed higher in 4 week,8 week and 12 week than SPF chickens on the same age. In kidney samples of ALV-J infected group only JUN gene expressed lower, other genes all expressed higher in 8 week. In clinical liver tumor tissues the β-catenin gene expressed higher; In clinical kidney tumor tissues LEF gene and WIF gene expressed higher. In clinical liver tumor tissues of MDV infected group, only TCF7 gene expressed lower, WIF gene was not detected. But in clinical kidney tumor tissues of MDV infected group TCF7 gene and WIF gene expressed higher. In conclusion, β-catenin gene and LEF gene expressed higher in most groups. WIF gene can’t be detected in liver tissues, but expressed higher in kidney tissues. These studies suggested that Wnt/β-catenin signaling pathway might play a role in avian tumor diseases. |
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