Cloning Expression And Biological Properties Of Porcine LIGHT And Carp GILT

LIGHT is the 14th member of tumor necrosis factor cytokine superfamily,playing a critic role in activation of dendritic cells,co-stimulating of T lymphocyte and inducing the apotosis of tumor.Pig is an important economic animal which can serve as an animal model organism for biomedical research and human diseases.For these applications,we isolated a LIGHT gene homologue from pig(designated PLIGHT).Firstly,we analyzed the cDNA sequence of pLIGHT.As other TNFSF members,pLIGHT is a type II transmembrance protein containing a short transmembrane segment,a potential cleave site,two cysteine residues and a C-termeinal TNF homology domain.In order to study the role of LIGHT in the immune system,we constructed a new fusion protein of the extracellular domain of pig LIGHT with the Fc fragment(named psLIGHT-Fc).Escherichia coli expression system is a low cost and fast expression system,so we construted a recombinant plasmid which named pET28a-psLIGHT-Fc.Then we transformed this recombinant plasmid into E.coli BL21(DE3)and obtained soluble psLIGHT-Fc protein,which were confirmed by SDS-PAGE and Western blot analysis.psLIGHT-Fc protein not only can combine with ProteinA column,greatly simplifying the purification process but also can quickly and conveniently to use flow cytometry and confocal laser scanning,method to detect the activity via the Fc tag combining with the corresponding antibody.Moreover,the MTT assays analysis indicated that psLIGHT-Fc protein could promote the proliferation of mouse splenic T cells together with anti-mouse CD3.These findings can provide some help for the study of the relationship between LIGHT and the immune system.In addition,a GILT homologue has been identified in mandarin fish(Siniperca chuatsi),and its biological activities have been characterized.Through reverse transcription PCR and rapid amplification of cDNA ends(RACE)strategies,we obtained the full-length cDNA of mGILT,which consists of 1008 bp with a 771 bp open reading frame,a 27 bp 5'-untranslated region(UTR)and a 3'-terminal UTR of 210 bp.We conducted bioinformatics analysis of mGILT through the Clustalw,MEGA4.0 and Protparam online tools.Meanwhile,real-time quantitative PCR(qPCR)analysis indicated that mGILT was predominantly expressed in the spleen of mandarin fish.The mGILT was efficiently expressed in Escherichia coli BL21(DE3)and confirmed by SDS-PAGE and Western blot analysis.Further study revealed that mGILT exhibit thiol reductase activity on IgG substrate.These results suggest mGILT could catalyze disulfide bond reduction and is highly likely to play an important role in the fish immune responses.