cDNA cloning, genomic structure, expression analysis and biological activity of porcine A Proliferation-Inducing Ligand (APRIL).
A Proliferation-Inducing Ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily. In this study, a novel cDNA has been isolated from pig spleen by homology cloning and 3'- and 5'-rapid amplification of cDNA ends (RACE) strategies and designates porcine APRIL (pAPRIL). The open reading frame (ORF) of this cDNA covers 756 bases, encoding 251 amino acids. The soluble part of pAPRIL shows 89% identity with its human counterpart at the level of the primary protein structure. The pAPRIL gene is approximately 2.1kb in size and comprises six exons and five introns. Southern blotting analysis indicated that the pAPRIL gene is a single copy gene. Real-time PCR analysis revealed that pAPRIL is constitutively expressed in various tissues. Recombinant His(6)-tagged psAPRIL protein was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. In vitro, purified recombinant psAPRIL protein co-stimulated the proliferation of porcine splenic B-cells in response to formalin-fixed Staphylococcus aureus Cowan 1 (SAC).
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, PR China.
Yan
Y
Shui
Zheng-Bing
ZB
Guan
Jia-Xin
JX
Zhang
Shuang-Quan
SQ
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D015139:Blotting, Southern / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005562:Formazans / Q000737:chemistry; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D013552:Swine / Q000235:genetics* / Q000276:immunology; D013778:Tetrazolium Salts / Q000737:chemistry; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13 / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D005562:Formazans; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13; C079782:MTT formazan; D012313:RNA