Caspase Recruitment Domain(CARD)of Swine BC L10 Impacts Its Expression And Cell Cycle

NF-κB pathway can activate or inhibit the expression of nucleus genes to transfer the external stimuli into the cell signal during the process of cell signal transduction. In the immune system, NF-κB plays a similar role in the controlling the pathway of antigen receptor signaling transduction as well as the regulation and induction of nucleus transcription factors. BCL 10 is an important functional gene involving in NF-κB pathway and its normal expression can promote the production of immune factors, enhance innate immunity and defence capacity. Conversely, the abnormal expression of BCL 10, such as over-expression, over-phosphorylation and nucleus expression will lead to abnormal expression of a series of cytokines, thereby affecting the physiological function of normal cells and even resulting in various diseases including lymphoma. It is well known that CARD domain, phosphorylation sites, and the C terminal sequence of BCL10 are involved in the fuctions of almost all of the protein. And the research of our group on swine BCL 10 was carried out based on the previous research.In a previous work of our group, full-length gene of swine BCL 10 was obtained by in silico cloning and coding sequence of this gene were cloned from swine spleen using specific primer. And its protein of prokaryotic expression was obtained and characterized. Scanning the expression profile of BCL 10 in vivo in swine indicated that the highest mRNA expression of swine BCL 10 gene was observed in spleen, followed by thymus, brain, and mandibular lymph node. Comparatively, there was only a trace of mRNA expression and no expression in kidneys.Our present study explored CARD domain-related functions of swine BCL 10 gene through the analysis of gene sequence and eukaryotic expression. And our results obtained in the present work are as follows:1. According to the characteristics of swine BCL10 gene, the possible sites related to nuclear expression was found through the prediction of the kinase action sites and the spatial structure prediction. And the vector pEGFP-B was successfully constructed for complete swine BCL10 gene, meanwhile the vector pEGFP-D(deleting swine BCL 10 gene in CARD structure domain) and pEGFP-P(deleting swine BCL 10 gene at Thr- Ser-Ser site) were constructed through site-mutant PCR method (large primers amplification method). 2. After the above three vectors were transfected into swine umbilical vein endothelial cells (SUVEC), the analysis of Western Blot showed that the fusion protein was expressed. Our analysis showed that TSS sites (Thr-Ser-Ser) involved in CARD domain of swine BCL 10 and interacted with other proteins, which are important sites of the gene. Meanwhile, deleting TSS sites will lead to nucleus expression of BCL 10, thus proving the function of TSS in swine BCL 10 gene.3. The analysis of cell cycle showed that CARD domain of swine BCL 10 would cause shortening of G 1 phase but prolonging of S phase and G 2/M phase, which indicates that domain structure affects DNA replication and cell mitosis, and excessive expression will lead to cell arrest in early stage of cell division, but will not lead to cell apoptosis. This lays a good foundation for swine BCL 10 function in cell signal transduction as well as diagnosis and treatment of disease such as lymphoma, etc.