High-efficiency Expression, Purification And Biological Activity Detection Of Domestic Horse SAPRIL/Fc And SBAFF/Fc Fusion Proteins

APRIL and BAFF,which play fundamental role in maturation,activiation and survival of both T lymphocytes as well as B lymphocytes,are important members belonging to the tumor necrosis factor superfamily(TNFSF).Other studies have shown that APRIL,BAFF were also involved in autoimmune disease and tumor immunity.In this study,overlap PCR was used to splice the soluble form of equine APRIL and BAFF to Fc fragment,and the splice product was subcloned into the pET28a expression vector.EcsAPRIL/Fc,ecsBAFF/Fc fusion proten with a theoretical molecular weight of 43.17kDa and 43.72kDa were high-efficiency expressed after transforming the recombinated plasmids into the ArcticExpress(DE3)host cells.Western Blot was applied to further identify the recombinated protein.Three-dimensional structure prediction showed that cytokine part and Fc part in the fusion protein could maintain their own space conformation,suggesting both of the two parts can function correctly.Results of non-reduced SDS-PAGE shown that the recombinated protein can form more complex polymer.Our results concluded that ecsAPRIL/Fc,ecsBAFF/Fc can bind to the receptors in mice B lymphocytes and promote the proliferation of B cells in a dose-depended manner after confocal laser scanning,flow cytometry analysis and CCK-8 assay.The high-efficiency expression,purification systerm of ecsAPRIL/Fc and ecsBAFF/Fc can be used as fundamental skills in futher applied research.Additionally,a TRAIL homologue,which was named ecTRAIL cloned from equine adipose-derived stem cell(ADSC)was for the first time reported in this study.TRAIL,as one of the key member in TNFSF,plays an important role in immunological surveillance and antiviral immunity.Results in our study revealed that the ORF of ecTRAIL was 870bp,encoding 289 amino acids.Secondary structure of ecTRAIL protein can be devided into three regions:the extracellular region,the transmembrane region and the intracellular region,which exhibited a classic type-? transmembrane protein structure similarly to other TRAILs.The amino acid sequence alignments and protein tertiary structure predictions showed that ecTRAIL shared high homology and similarity of molecular structure,suggesting ecTRAIL may be involved in immunoregulatio.Based on the pro-solubility of SUMO-tag,SUMO-taged fusion protein combined with ecsTRAIL was expressed as a soluble form.The fusion protein SUMO-ecsTRAIL can bind to the TRAIL receptors in HeLa cells and suppress the proliferation of HeLa cells in a doze-depended pattern in vitro.