Palanisamy, Rajesh

English

University of Maine

CHANNA STRIATUS TNFR-1: MOLECULAR CLONING, CHARACTERIZATION AND GENE EXPRESSION  Rajesh Palanisamy, Jesu Arockiaraj* Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, Tamil Nadu, India. ABSTRACT Tumor necrosis factor receptor (TNFR) superfamily entails in various biological functions including cell proliferation, differentiation, survival and providing co-stimulatory signals for programmed cell death or apoptosis. In this report, we have identified and characterized TNFR-1 from Channa striatus (Cs) at molecular level. In silico analyses revealed that the CsTNFR-1 cDNA sequence was 1200 bp in length, which includes an open reading frame of 1197 bp that encoded a putative protein of 399 amino acids (45 kDa). It contains three ‘Cys’ rich domains (CRDs) at CRD141-46, CRD279-118 and CRD3120-159 in the extracellular region, also it possessed a putative TRAF6-binding site in the cytoplasmic tail. In addition, CsTNFR-1 cytoplasmic region contained a death domain between 297 and 388, which is important for apoptosis induction. It shared a maximum identity with its homologous from Paralichthys olivaceus (82%). Real-time PCR findings revealed that CsTNFR-1 was expressed predominantly in head kidney, further it is up-regulated upon fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infection. Overall, the gene expression results indicated that CsTNFR-1 involvement in the innate immune response against pathogenic infections. The recombinant CsTNFR-1 protein was over-expressed and purified using an Escherichia coli expression vector system. The purified recombinant CsTNFR-1 derivative binds with TNF-α at high affinity and inhibits its cytotoxic activity in vitro in a dose dependent manner. CsTNFR-1 was also analyzed for respiratory burst activity upon two different ROS inducers such as opsonized zymosan and phorbol 12-myristate 13-acetate (PMA). The results showed that the recombinant CsTNFR-1 protein alone did not produce any ROS, but upon induction with opsonized zymosan, it considerably increased the ROS production in a concentration dependent manner. Overall, the findings showed the potential involvement of CsTNFR-1 in the pathogen-induced inflammatory process of C. striatus.   KEYWORDS  Channa striatus; Tumor necrosis factor receptor 1; TNF-α inhibition activity; Cytotoxic activity; Respiratory burst activity.  * Corresponding author. Tel.: +91 44 27452270; fax: +91 44 27453903.     E-mail address: jesuaraj@hotmail.com (J. Arockiaraj). 

CHANNA STRIATUS TNFR-1: MOLECULAR CLONING, CHARACTERIZATION AND GENE EXPRESSION

https://digitalcommons.library.umaine.edu/cgi/viewcontent.cgi?article=1002&amp;context=isfsi

CHANNA STRIATUS TNFR-1: MOLECULAR CLONING, CHARACTERIZATION AND GENE EXPRESSION

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University of Maine