Construction Of Suvec Line Stably Expressing Porcine MNSFβ And Investigation Of Its Pro-apoptosis Function

Monoclonal non-specific suppressor factor β(MNSFβ), also know as a ubiquitously expressed non-specific immunosuppressive factor, is also a cell apoptosis regulates factor and potential tumour suppressor factor that played an vital role of immunoregulation and cell apoptosis In recent years, MNSFβ gene has been studied in mouse. However, study on porcine MNSFβ has been rarely reported. Pigs are more optimal animals used as medical models than mice. Therefore, the study on pMNSFβ gene will be helpful to study human MNSFβ gene and related diseases. In this study, we studied the function of pMNSFβ and mainly focused on interactions with porcine BCL-G(pBCL-G) and its enhancement to apoptosis. The results were shown as follows.1. By transient transfection and selection with G418, a clone of SUVEC cells stably expressing GFP-pMNSFβ protein were obtained respectively. By fluorescence analysis and western blot analysis, the expression of GFP-pMNSFβ protein was detected in the clone of cells. These results indicate that we established SUVEC line stably expressing pMNSFβ successfully.2. Co-immunoprecipitation assay showed that pMNSFβ protein could interact with pBCL-G protein.3. By light microscopy analysis, compared with control group, a series of typical apoptotic features were observed in cells stably expressing GFP-pMNSFβ protein induced by staurosporine(STS), such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells. By staining with Hoechst 33342 and fluorescence analysis, compared with control group, chromatin condensation and nuclear fragmentation were observed in cells stably expressing GFP-pMNSFβ protein induced by STS.4. Annexin V-PE/PI staining and flow cytometry analysis showed that stably expressed pMNSFβ protein significantly enhanced apoptosis induced by STS. pMNSFβ and pBCL-G protein synergistically enhanced apoptosis induced by STS.