Research Article

Construction of pCMV-myd88 and pCMV-traf6 Eukaryotic Expression Vectors in Zebrafish (Danio rerio)  

Xing Xu1,2 , Zirui Wang1,2 , Heyi Liu1,2 , Ying Zhang1,2 , Jianfeng Ren1,2 , Weiming Li3 , Qinghua Zhang1,2
1 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai, 201306, China
2 National Pathogen Collection Center for Aquatic Animals, Shanghai, 201306, China
3 Department of Fisheries and Wildlife, Michigan State University, East Lansing, 48824, China
Author    Correspondence author
International Journal of Aquaculture, 2020, Vol. 10, No. 2   
Received: 14 Apr., 2020    Accepted: 16 Apr., 2020    Published: 16 Apr., 2020
© 2020 BioPublisher Publishing Platform
This article was first published in Genomics and Applied Biology in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Both myeloid differentiation primary response gene 88 (MyD88) and TNF receptor associated factor 6 (TRAF6) are key adaptor molecules in the Toll-like receptors family (TLR). Zebrafish is a unique model organism for studying the innate immune response. In order to construct eukaryotic expression vectors of MyD88 and TRAF6, and use them to study the immune response mechanism, we cloned the full-length CDS sequence of the coding region of zebrafish myd88 and traf6 genes in this study. The full length of zebrafish myd88 and traf6 gene CDS is 855bp and 1629bp, respectively. Structural analysis showed that there are two conserved domains of zebrafish MyD88 including DD domain and TIR domain; four conserved domains of TRAF6 including RING domain, two zf domains, coiled-coil and MATH. And they have high amino acid sequence identity with other species. Phylogenetic tree analysis found that the zebrafish myd88 or traf6 gene has high structural homology and closely related to teleost fish, indicating that they are consistent with their evolutionary status. Then the CDS sequence of myd88 and traf6 genes were constructed to the expression vector of pCMV-Tag2B. Zebrafish pCMV-myd88 and pCMV-traf6 eukaryotic expression vectors were successfully cloned by double enzyme digestion. In order to verify the biological function of these two eukaryotic expression vectors, we performed NF-κB reporter gene verification in HEK293T cell. After overexpression of myd88 and traf6 gene, the transcriptional activity of nfκb1 in zebrafish NF-κB family is significantly increased, about 2.5 and 8 times, respectively, contract to that of the control group. In view of the important role of MyD88 and TRAF6 in innate immune function, our results provide a powerful research tool for the future study on the innate immune signal transduction process of zebrafish.

Keywords
Zebrafish; pCMV-myd88; pCMV-traf6; Eukaryotic expression vectors
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International Journal of Aquaculture
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. Xing Xu
. Zirui Wang
. Heyi Liu
. Ying Zhang
. Jianfeng Ren
. Weiming Li
. Qinghua Zhang
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