Molecular structure, expression, and function analysis of BAFF gene in Chinese sucker, Myxocyprinus asiaticus
B cell activating factor (BAFF), belonging to the tumor necrosis factor superfamily (TNFSF), is a critical cytokine for B cell survival and immunoglobulin secretion. Here, the BAFF gene of Chinese sucker ( Myxocyprinus asiaticus ) ( MaBAFF ) was cloned using RT-PCR and RACE (rapid amplification of c...
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Veröffentlicht in: | Fish physiology and biochemistry 2021-04, Vol.47 (2), p.225-238 |
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Zusammenfassung: | B cell activating factor (BAFF), belonging to the tumor necrosis factor superfamily (TNFSF), is a critical cytokine for B cell survival and immunoglobulin secretion. Here, the BAFF gene of Chinese sucker (
Myxocyprinus asiaticus
) (
MaBAFF
) was cloned using RT-PCR and RACE (rapid amplification of cDNA end) techniques. The open reading frame (ORF) of
MaBAFF
encodes a 272-amino acid protein containing a transmembrane domain, a TNF family signature, and a putative furin protease cleavage site as seen in BAFFs from other species. Tissue expression profiles of
MaBAFF
determined by absolute and relative quantification of real-time quantitative PCR (qPCR) showed that
MaBAFF
is widely distributed in various tissues, with the highest expression in spleen.
MaBAFF
can be detected during fertilized egg period by RT-PCR. Upon induction by
A. hydrophila
, the expression of
MaBAFF
was up-regulated in spleen from 48 to 72 h, and the expression of BAFF and IgM all reached a peak at 48 h in head kidney. The soluble BAFF gene (
MasBAFF
) had been cloned into pET30a. SDS-PAGE and Western blotting analysis confirmed that the His-
MasBAFF
was efficiently expressed in
Escherichia coli
Rosset (DE3). CCK-8 assay indicated that the
MasBAFF
recombinant protein (200 ng/ml) could prolong the survival of peripheral blood leukocytes. Based on ELISA screening and Western blotting, monoclonal antibody 1-F2A3 against recombinant
MasBAFF
was selected and used for immunohistochemistry, which showed that BAFF-positive cells were detected in spleen and head kidney. Our results raise the possibility that
MaBAFF
may be useful to enhance immune efficacy in Chinese sucker disease defense. |
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ISSN: | 0920-1742 1573-5168 |
DOI: | 10.1007/s10695-020-00906-5 |