The Construction And Application Of Human Lectin Microarray

Lectin has extraordinary advantages as a kind of protein which can speciallyidentify different sugar chain under the natural state, especially in glycosylation analysisof cells. The aim of this study is to construct a human lectin microarray and apply toresearch the relationship of migration of cancer cells and human lectins. This study willinvestigate the function of human lectins at a system level and the results will give thedirection for the breast cancer diagnose and treatment.The study collected106ORFs of human lectins. Lectin Entry clones wererecombined with pEGH-A plasmid of the yeast by LR reaction of Gateway technologyand yeast were induced to express the recombinate proteins. Zirconium balls wereadded to the tube and the yeast were lysised by severe shock, and those recombinateproteins with GST tag were riched by glutathione affinity matrix. The synchronousmethod of lysis and purify could improve the efficiency of purification and keep theiractivity of membrane proteins. These ORFs constructed a yeast expression library werefrom a yeast expression library of all human proteins and original Entry clones. Therewere69speicies in the yeast expression library matched with the database online,51ofthem were successfully expressed by the yeast.24Entry clones were transfected intoyeast,23of them were expressed successfully.After compared different substrate, PSH-OP substrate was chosen to constructhuman lectin microarray because it is the best one in the signal intensity, SNR and so on.Each microarray had12blocks, and each block had13rows and18columns,74lectinproteins,2negative controls and1positive control were arranged on a block, eachprotein had3repeats. The availability of the human lectin microarray was detected byquality control, and it worked.Triple-negative breast cancer line (MDA-MB-231）and cell lines（SCP2，SCP4，SCP6，SCP28and4175）which had different migration ability and addiction to diverseorgans,6lectin（sGalNAc-T6、galectin-1、galectin-2、galectin-7、galectin-8、ERGIC-53）were found those could specially bound with sugar. All of them can be further used tostudy breast cancer.Collected coding sequences were transfected into yeast. Proteins were printed onthe chip after they were isolated and purified. It is a platform to analyze the change ofthe carbohydrate on the breast cancer cells. It makes sense for the primary diagnose of breast cancer.