Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in rainbow trout ( O
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ORIGINAL PAPER
Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in rainbow trout (Oncorhynchus mykiss) with implications for GILT in innate immune response Meng Liu & Hongyan Liu & Xiaocui Guan & Hongxin Ai & Haitao Wu & Ping Liu & Wei Gu & Shuangquan Zhang
Received: 20 November 2012 / Accepted: 26 March 2013 / Published online: 5 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The interferon-γ-inducible lysosomal thiol reductase (GILT) has been demonstrated to play an important role in the processing and presentation of MHC class IIrestricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a rainbow trout cDNA (designated as rGILT) was cloned and identified from Oncorhynchus mykiss. The open reading frame of rGILT consists of 759 bases encoding a protein of 253 amino acids with an estimated molecular mass of 28.23 kDa and a theoretical isoelectric point of 4.94. The rGILT exhibited a characteristic GILT signature sequence CQHGX2ECX2NX4C and CXXC motif. Phylogenetic analysis suggested that rGILT had been derived from a common ancestor with other GILT proteins. RT-PCR results showed that rGILT and rIFN-γ (rainbow trout IFN-γ) mRNA was expressed in a tissue-specific manner and obviously up-regulated in splenocytes and the cells from head kidney after induction with LPS. Recombinant rGILT fused with His6 tag was efficiently expressed in M. Liu : H. Liu : X. Guan : H. Ai : H. Wu : P. Liu : W. Gu : S. Zhang Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210023, China M. Liu : H. Liu : X. Guan : H. Ai : H. Wu : P. Liu : S. Zhang (*) Jiangsu Key Laboratory for Aquatic Crustacean Diseases, Life Sciences College, Nanjing Normal University, Nanjing 210023, China e-mail: [email protected] W. Gu (*) Pharmacy College, Nanjing University of Chinese Medicine, Nanjing 210023, China e-mail: [email protected]
Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Further study revealed that rGILT was capable of catalyzing the reduction of the interchain disulfide bonds from intact IgG. This study shows that rGILT may be involved in the immune response to bacteria challenge and maintain first line of innate immune defense at basal level in O. mykiss. It also provides the basis for investigating on the role of GILT using O. mykiss as an animal model for related studies. Keywords Oncorhynchus mykiss . rGILT . Phylogenetic analysis . Real-time PCR . Lipopolysaccharide (LPS) . Thiol reductase activity
Introduction The interferon (IFN) system is the cellular defense mechanism that acts against viral infections by inhibiting multiple steps of viral replication, activating macrophages and increasing antigen-presentation process (Cebulla et al. 1999; Jones et al. 1982). Antigen processing and presentation pathways are central to the generation of immune responses. Within the MHC class II antigen processing pathway, the reduction of disulfid
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