Human Soluble Proliferation-inducing Ligand Expression, Purification And Identification Of Its Activity And Antibody Preparation

A proliferation-inducing ligand(APRIL) is a new member of the TNF family. Among the divergent TNF ligand family, APRIL is most closely related to BAFF(B cell activating factor) .In this paper, on the one hand,the total RNA was isolated from the human peripheral blood and its cDNA was successfully cloned into the pET-30a vector. The cytokine was expressed and after denaturation and renaturation its active form was obtained which was confirmed by the proliferation assay. On the other hand, the protein was used as antigen to immunize the rabbit and the polyclonal antibody was obtained from its blood. Then the polyclonal antibody was purified by the Protein-A affinity column and analyzed by SDS-PAGE, Western blotting and ELISA assays. The design of the experiments and the results were as followings:1. Expression, purification and activities assay of hsAPRIL protein.The cDNA gene of hsAPRIL was obtained by RT-PCR from the human peripheral blood and then cloned into the BL21(DE3) to express the target protein. The hsAPRIL was purified by immobilized Ni -IDA affinity chromatography. After denaturation the purified protein obtained its biological activity which was verified by the tumor cell proliferation assay.2. Preparation and purification of polyclonal antibody of hsAPRIL The experiment rabbit was immunized by hsAPRIL every 7days andafter the third immunization the blood was got form its heart and itsspecificity and titer were analyzed by Western blotting and ELISArespectively.