Molecular Cloning, Recombinant Expression, and Funtional Characterization of APRIL (TNFSF13) in Cat (Felis catus)
2016
Liu, Hong-Zhen | Song, Ren | Zhang, Jia-Xin | Li, Jian-Feng | Gu, Wei | Zhang, Shuang-Quan
A proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn ¹²⁴), and two conservative cysteine residues (Cys ¹⁹⁶ and Cys ²¹¹). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species.
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