[Eukaryotic expression of human LIGHT-Fc chimeric molecule and its anti-tumor activity].
OBJECTIVE
Construct eukaryotic expression plasmid of hLIGHT-Fc gene and express hLIGHT-Fc fusion protein with high biological activity.


METHODS
Utilizing PCR to clone the human LIGHT cDNA from a normal human activated T cells cDNA library phAD. CAD, then using SOE (splicing by overlap extension) technique to fuse CD137 signal peptide gene into LIGHT extramembrane domain encoding sequence, inserting this recombinated LIGHT cDNA with human IgGI Fc cDNA into the eukaryotic expression plasmid pcDNA3, expressed it transiently in 293T cells, and purified by recombinated protein A affinity chromatography column, at last we checked its Mr, purity and anti-tumor activity.


RESULTS
LIGHT-Fc gene's ORF is right and coincident with we expected, then its expression was confirmed by ELISA and SDS-PAGE, and we proved that the purified LIGHT-Fc protein could inhibit the growth of some cultured tumor cell lines by MTT.


CONCLUSIONS
we got a purified recombinated protein of LIGHT-Fc with anti-tumor activity and the anticipated Mr, thus lay a foundation for further works on LIGHT such as its role in immune homeostasis,the mechanism of its receptors' signal transduction, et al.
Huang
Gang
G
Department of Molecular Genetics, Third Military Medical University, Chongqing 400038, China. immuno02@mail.tmmu.com.cn
Bai
Yun
Y
Jiang
Man
M
Li
Wan-ling
WL
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000494:pharmacology
D000970:Antineoplastic Agents; D007141:Immunoglobulin Fc Fragments; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C505914:TNFSF14 protein, human; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D014409:Tumor Necrosis Factor-alpha