Preparation Of Galectin-3 And The Effect Of Pectin On The Interaction Between Galectin-3 And T Cells

Galectin-3 is the only chimeric member among the galectins family. Its molecular weight is 30～35kD.Galectin-3,as a tumor-related protein, plays an important role in cell adhesion, proliferation, apoptosis, angiogenesis and mRNA aplicing by interacting with intracellular and extracellular molecules.It is closely related to tumor growth and metastasis.Pectin is a type of acidic polysaccharide and has anti-tumor and immuno-modulation activities. Studies have shown that pectin may exert its anti-tumor effect via interacting with galectin-3.In this thesis, I successfully constructed the expression plasmid of galectin-3, optimized expression conditions, purified galectin-3 proteins, checked the activities of galectin-3,and finally studied the effect of pectin on galectin-3-T cell interactions.Construction of galectin-3 expression plasmid:full length galectin-3 cDNA was produced by PCR using the cloning plasmid pOTB7-Gal-3 as the template. The expression plasmid pET-22b-(+)-Gal-3 was constructed and sequence checked by agarose gel electrophoresis and DNA sequencing.Optimization of galectin-3 expression conditions:galectin-3 expression was studied under different temperature and concentrations of IPTG using E. coli expression system, and measured by SDS-PAGE and Western-blotting. The results showed that 25℃and 218.5μM IPTG were the best conditions for protein expression.Extraction and purification of galectin-3 proteins:asialofetuin was coupled to sepharose-4B beads. Galectin-3 proteins were extracted and purified by affinity chromatography using asialofetuin-sepharose-4B column.Checking the activities of galectin-3:T-cell aggregation, membrane adsorption and cell apoptosis assays were used to check the activities of galectin-3 proteins.The results showed that the obtained galectin-3 proteins had anticipated activities.Testing the effect of pectins on the interactions between galectin-3 and T cells: three pectin samples-Galactan, RGI-4 and MCP were tested for their effects on galectin-3-related T-cell aggregation, membrane adsorption and cell apoptosis.The results showed that RGI-4 significantly inhibited the binding of galectin-3 to T-cells.