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A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
Published on: October 30, 2012
Molecular cloning, characterization and expression profiles of CD2AP in Nile tilapia (Oreochromis niloticus)
Yuan Li1, Bei Wang2, Zhi-Wen Wang1
1Shenzhen Institute of Guangdong Ocean University, Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen, 518120, Guangdong, China; Fisheries College of Guangdong Ocean University, Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Zhanjiang, China.
Abstract:
The interaction between CD2-associated protein (CD2AP) and CD2 plays a vital role in lymphocyte adhesion and T cells activation in mammals. In this study, a CD2AP gene (GenBank accession number: MK579862; designated as On-CD2AP) was identified from tilapia (Oreochromis niloticus). Sequence analysis showed that On-CD2AP protein shares high similarity with mammals, including three Src homology 3 (SH3) domains, a section of poly proline motif and a coiled coil region. Transcription levels of On-CD2AP were detected in nine tissues of healthy Nile tilapia, and the highest expression levels were detected in the spleen and gill. On-CD2AP were significantly up-regulated in thymus, head kidney and brain after infected by Streptococcus agalactiae, as well as in head kidney leukocytes (HKLs) with LPS and LTA stimulation. Moreover, a section conserved pro-rich motif that are responsible for binding of CD2 to CD2AP were found in the CD2 cytoplasmic sequence of Nile tilapia (On-CD2C). A weak interaction between On-CD2AP and On-CD2C was proved by yeast two-hybrid assay. In addition, the recombinant proteins of CD2AP-His (rOn-CD2AP-His) and GST-CD2C (GST-rOn-CD2C) were obtained through prokaryotic expression system. His pull-down assay showed that rOn-CD2AP-His and GST-rOn-CD2C could bind to each other. These findings indicate that CD2AP is crucial in immune response during S.agalactiae infection, and the mechanism of interaction between CD2AP and CD2 is conservative in Nile tilapia.
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