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| Molecular Cloning, Expression and Functional Analysis of TNFSF13b (BAFF) Gene in Goldfish, Carassius auratus |
| PEI Li-li1, HUANG Fang2, LI Gui-ting2, PANG Shu-ying2, REN Wen-hua2 |
1. Kangda College of Nanjing Medical University, Lianyungang 222000, China;
2. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China |
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Abstract Objective: To clone the goldfish B cell activating factor (BAFF) gene and construct soluble BAFF expression vector, and investigate the effect of the expression product on goldfish lymphocytes B cells. Methods: The full-length cDNA of BAFF from the goldfish (designated CaBAFF) was cloned using RT-PCR techniques. Using Rapid amplification of cDNA ends (RACE) to obtain the full-length cDNA of CaBAFF including the 5' and 3' untranslated regions (UTRs), and its soluble BAFF gene (designated CasBAFF) was linked with SUMO, efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by Western blot analysis. Laser scanning confocal microscopy analysis showed that SUMO-CasBAFF could bind to the surfaces of goldfish lymphocytes B cells. In vitro, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay analysis if the purified Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. Results: The goldfish BAFF was successfully constructed and obtained a soluble Nus-His-CasBAFF protein. In vitro, the soluble Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. Conclusions: The purified soluble Nus-His-CasBAFF protein could induce the survival/proliferation of goldfish splenic B cells. These results about goldfish BAFF gene would provide a basis for understanding the characteristics of the immune system of the goldfish.
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Received: 23 June 2016
Published: 25 December 2016
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| [1] Schneider P, MacKay F, Steiner V, et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med, 1999,189:1747-1756.
[2] Daridon C, Youinou P, Pers J O. BAFF, APRIL, TWE-PRIL:Who's who?. Autoimmunity Reviews, 2008, 7(4):267-271.
[3] Mukhopadhyay A, Ni J, Zhai Y, et al. Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J Biol Chem, 1999,274:15978-15981.
[4] Moore P A, Belvedere O, Orr A, et al. BLyS:member of the tumor necrosis factor family and B lymphocyte stimulator. Science, 1999,285:260-263.
[5] Locksley R M, Killeen N, Lenardo M J. The TNF and TNF receptor superfamilies:integrating mammalian biology. Cell, 2001,104:487-501.
[6] Pieper K, Grimbacher B, Eibel H. B-cell biology and development. Journal of Allergy and Clinical Immunology, 2013, 131(4):959-971.
[7] Wang P H, Wan D H, Pang L R, et al. Molecular cloning, characterization and expression analysis of the tumor necrosis factor (TNF) superfamily gene, TNF receptor superfamily gene and lipopolysaccharide-induced TNF-α factor (LITAF) gene from Litopenaeus vannamei. Developmental & Comparative Immunology, 2012, 36(1):39-50.
[8] Mackay F, Browning J L. BAFF:a fundamental survival factor for B cells. Immunology, 2002,2:465-475.
[9] Moisini I, Davidson A. BAFF:a local and systemic target in autoimmune diseases. Clin Exp Immunol, 2009,158:155-163.
[10] Ng L G, Mackay C R, Mackay F. The BAFF/APRIL system:life beyond B lymphocytes. Mol Immunol, 2005,42:763-772.
[11] Schneider P, MacKay F, Steiner V, et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med, 1999,189:1747.
[12] Thompson J S, Bixler S A, Qian F, et al. BAFF-R, a newly identifed tnf receptor that specifically interacts with BAFF. Science, 2001,293:2108-2111.
[13] Wu H T, Yan X M, Hu Y L, et al. Expression, characterization of recombinant human soluble BAFF secreted from CHO cell. Mol Biol, 2009,43(1):76-81.
[14] Liang Z, Kong Y, Luo C, et al. Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danin rerio). Fish Shellfish Immunol, 2010,29(2):233-240.
[15] Ai H, Shen Y, Min C, et al. Molecular structure, expression and bioactivity characterization of TNF13B (BAFF) gene in mefugu, Takifugu obscurus. Fish Shellfish Immunol, 2011,30(6):1265-1274.
[16] Dan W B, Guan Z B, Zhang C, et al. Molecular cloning, in vitro expression and bioactivity of goose B-cell activating factor. Vet Immunol Immunopathol, 2007,118:113-120.
[17] Liu H, Zhang J, Li J, et al. Molecular structure, distribution, and immunology function of TNFSF13B (BAFF) in Nile tilapia (Oreochromis niloticus). Fish & Shellfish Immunology, 2016, 51:240-250. |
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