| LIGHT, also named TNFSF14 or HVEM-L, is a recently identified member of the TNF cytokine superfamily.It is a 29-kD type II transmembrane glycoprotein expressed mainly by activated T cells and immature DCs . LIGHT can initiate diverse biological functions depending on the receptor expression profile of target cells and the cytokines secreted by T cells. There are three receptors of LIGHT: LTβR, expressed on stromal cells, non-lymphoid hematopoietic cells and many tumor cells; Herpes virus entry mediator (HVEM), expressed constitutely on T, B, and other hematopoietic cells; TR6 (also called DcR3) , expressed mainly by several normal human tissues such as the stomach, spinal cord, colon, lymph node, spleen,and by tumors from lung and colon,expressed weakly in the thymus and not expressed in peripheral blood lymphocytes .It is also a secreted decoy receptor to FasL.They all belong to the TNFR superfamily. The interaction between LIGHT and TR2 can costimulate T cell proliferation in a CD28-indepedently manner, preferly induce CTL response and Th1-type cytokines secretion such as IFN-γand GM-CSF;while the interaction between LIGHT and LTβR can induce the apoptosis of certain tumor cell lines in particular circumstances,the death signaling is transduced through TRAFs .TR6 acts as a modulator,can interfere or block the biological effects when LIGHT binds to HVEM or LTβR ,thus modulate the immune homeostasis,but some tumors also express TR6 as a means of immune evasion.More and more datas have suggested LIGHT played a pivotal role during the process of lymphotissue-organgenesis,tumorimmunity, autoimmunity,transplantation rejection and antivirus immunity.Beacuse of its extraordinary costimulatory and cytolytic activity, LIGHT has showed a strong potential in tumor therapy and other fields, it soon becomes a promising new star in the field of immunology. To further explore its detailed mechanism of antitumor effects and other biological functions, it is very necessary to get a soluble LIGHT protein,therefore we plan to clone a full length human LIGHT cDNA,,then constructe a hLIGHT-Fc chimeric molecule in eukaryotic expression vectors,at last get the hLIGHT-Fc fusion protein with highly activity through eukaryotic expression and affinity chromatography.It means much:At one hand,when this fusion protein binds to its receptors in vivo,may induce the apoptosis of tumor cells directly,as well as costimulate T cells activation to produce CTL responses and Th1-type cytokines secretion,which are very important for tumor immunotherapy,as the time is mature, this fusion protein can be developed as a biological agent;At the other hand,it can be used as a powerful tool to explore the subtle role of LIGHT in T cell homeostasis,the kinetics and the signaling pathway after LIGHT binds to its appropriate receptors.To get this goal,we conducted the following works:1. The construction of hLIGHT-Fc eukaryotic expression plasmids⑴ Utilizing PCR to clone the total length human LIGHT cDNA from a normal human activated T cells cDNA library phAD.CAD , confirming its open reading frame was the same as the public sequence of LIGHT in Genebank by DNA sequencing. ⑵ For the purpose of LIGHT's expression on the surface of mammal cells,inserting LIGHT cDNA into the eukaryotic expression plasmids:pCI-neo and pcDNA3 at EcoRI site.⑶ Using SOE(splicing by overlap extension) technique to fuse CD137 signal peptide gene into LIGHT extramembrane domain encoding region,theninserting this recombinated LIGHT cDNA and human IgG1 Fc cDNA into the eukaryotic expression plasmids:pCI-neo and pcDNA3, confirming by DNA sequencing.2. The expression and purification of hLIGHT-Fc fusion protein⑴ Expressing in mammal cells by liposome transfection, proving that the fusion protein was expressed in supernatant by sandwich-ELISA⑵ Purifing the fusion protein by recombinated protein A affinity chromatography column,then examining its molecular weight and purity by SDS-PAGE,we found this fusion protein's MW coincided wit...
|
PDF Full Text Request