Molecular Cloning, Recombinant Expression, and Funtional Characterization of APRIL (TNFSF13) in Cat (felis catus).
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.
a Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College , Nanjing Normal University , Nanjing , China.
Hong-Zhen
HZ
Liu
a Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College , Nanjing Normal University , Nanjing , China.
Ren
R
Song
a Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College , Nanjing Normal University , Nanjing , China.|b Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education , Nanjing Forestry University , Nanjing , China.
Jia-Xin
JX
Zhang
c Institute of Aging Research, School of Medicine , Hangzhou Normal University , Hangzhou , Zhejiang Province , China.
Jian-Feng
JF
Li
d College of Pharmacy , Nanjing University of Traditional Chinese Medicine , Nanjing , China.
Wei
W
Gu
a Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College , Nanjing Normal University , Nanjing , China.
Shuang-Quan
SQ
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects; D001483:Base Sequence; D002415:Cats / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology
D011994:Recombinant Proteins; D053300:Tumor Necrosis Factor Ligand Superfamily Member 13
B-cells proliferation; MTT assays; cat APRIL; in vitro expression; real-time quantitative PCR