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Molecular structure, expression, and function analysis of BAFF gene in Chinese sucker, Myxocyprinus asiaticus

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Abstract

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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Data availability

All data included in this study are available upon request by contact with the corresponding author.

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Acknowledgments

We are grateful for the technique support provided by Professor Deshou Wang at South west University.

Funding

This work was supported by grant CX2018097 from the Innovation Projects of Chongqing Overseas Students’ Innovation Support Plan, grant cstc2020jcyj-msxmX1045 from the Chongqing Natural Science Foundation of China, and grant 31972776 from the National Natural Science Foundation of China.

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Authors and Affiliations

  1. Key laboratory of Eco-environment in the Three Gorges Reservoir Region of Ministry of Education, School of Life Sciences, Southwest University, No. 1 Tiansheng Road, Beibei District, Chongqing, 400715, China

    Jiaojiao Zhang, Yujin Li, Xia Wu, Ruonan Zhong & Xiaoping Zhang

  2. Key laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, 400715, China

    Jing Wei & Zhijian Wang

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  1. Jiaojiao Zhang
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  2. Yujin Li
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  3. Xia Wu
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  4. Ruonan Zhong
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  5. Jing Wei
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  7. Xiaoping Zhang
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Contributions

JZ, YL, and XW conducted experiments and analyzed the data; RZ contributed to animal preparation; JZ and YL prepared the original manuscript draft; JW and ZW reviewed and edited the manuscript; XZ designed the whole experiment and reviewed the manuscript.

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Correspondence to Xiaoping Zhang.

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The authors declare that they have no conflict of interest.

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All animal experiments follow the laboratory animal welfare ethics principle with a permission number IACUC_20171212_01.

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Zhang, J., Li, Y., Wu, X. et al. Molecular structure, expression, and function analysis of BAFF gene in Chinese sucker, Myxocyprinus asiaticus. Fish Physiol Biochem 47, 225–238 (2021). https://doi.org/10.1007/s10695-020-00906-5

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