Rainbow Trout γ-IFN-induced Lysosomal Thiol Reductase And LIGHT (TNFSF14) Cloning, Expression And Biological Activity

The interferon-y-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play an important role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. LIGHT is a member of TNF super family, and plays important roles in reducing the apoptosis of tumors, con-stimulate of T lymphocyte, the development of lymphoid structure, and inducing the maturation of DCs. The nucleotide sequences, the amino acid sequences, tissue distribution and some biological activity of the two genes were analyzed, by means of molecular biology techniques such as Real-time PCR, prokaryotic expression and bio-informatics, etc. The results were summarized as follows:1The cloning, expression and biological activity of rainbow trout GILTIn this study, a rainbow trout cDNA (designated as rGILT) was cloned and identified from Oncorhynchus mykiss. The open reading frame (ORF) of rGILT consists of759bases encoding a protein of253amino acids with an estimated molecular mass of28.23kDa and a theoretical isoelectric point of4.94. 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 mRNA was expressed in a tissue-specific manner and obviously up-regulated in splenocytes and the cells from kidney after induction with LPS. And the expression of rIFN-γ had a similar trend after LPS stimulation. Recombinant rGILT fused with His6tag was efficiently expressed in 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 Oncorhynchus mykiss. It also provides the basis for investigating on the role of GILT using Oncorhynchus mykiss as an animal model for related studies.2The cloning, soluble protein expression and tissue quantitative analysis of rainbow trout LIGHT In this study, the LIGHT gene of717bp was amplified from rainbow trout (Oncorhynchus mykiss) spleen by RT-PCR and conducted phylogenetic analysis. The results of real-time PCR revealed that rLIGHT mRNA was expressed in various tissues and elevated in immune organs. The recombinant vector pETSUMO-rsLIGHT (rainbow trout soluble LIGHT) was also bulided and soluble recombinant protein was purified in vitro.