The Identification And Application Of Plasmatocyte' Specific Marker Bmintegrin ?2

The integrin is a superfamily of transmembrane protein which were composed of a and ? subunits.In mammalian,integrins triggers a large variety of signal transduction events such as adhesion,proliferation,survival or apoptosis,shape,polarity,motility,haptotaxis,gene expression,and differentiation.The intemin also play a very important role in the insects encapsulation-one kind of cell-mediated immunity.Integrin genes can also be specific markers for one type of cell,and as a molecular markers for it.Silkworm is the typical model insects,and all the tissues are surrounded by the circulating hemolymph.According to their different morphology,five types of hemocytes have been identified in silkworm,Bombyx mori:prohemocytes,plasmatocytes,granulocytes,spherulocytes,and oenocytoids.The study of hemocytes in circulatory system and HPO in vitro has been well investigated,but precise lineage relationships among different hemocyte types remain unclear.In the present study,we found and characterized a plasmatocyte specific marker Bmintegrin ?2 based on the silkworm database.By the expression of Bmintegrin ?2,the sources of plasmatocyte were investigated.The results are as follows:1.Full length gene cloning of Bmintegrin ?2 and analysisWe find Bmintegrin ?2 was specifically and highly expressed in hemocytes by searching the silkworm genome database,We cloned the Bmintegrin ?2 cDNA including the complete ORF sequence through the known sequence data and RACE.The sequence alignment and The phylogenetic tree showed that Bmintegrin ?2 was very and was closer to the ?1 of Manduca sexta.We predict the Bmintegrin ?2 has the Similar function with it.2.The mRNA expression pattern of Bmintegrin ?2 in silkworm:The Real-time PCR showed that Bmintegrin ?2 was specifical highly expressed in hemocytes.Bintegrin ?2 expressed from 7th day of embryo stage and maintained a high expression level untill the end of embryo stage.It suggests that the plasmatocyte begin to be produced from precursor cells on 7th day of embryo stage and exist in circulatory hemolymph.The expression level of Bmintegrin ?2 maintained a high levels the whole larva stage and reached the top at L5D6.3.Bmintegrin ?2 polyclonal antibody preparation and detectionA specific fragment of Bmintegrin ?2 had been chosed,and been cloned into prokaryotic expressional vector PET32a.This recombinant plasmid was induced to expressed successfully.After the proper inducing condition was determined,The available polyclonal antibody have been prepared.The results proved that antiserum specifically recognized the Bmintegrin ?2 fusion protein.Using antiserum for immunofluorescence,we found that the Bmintegrin ?2 can specifically recognize plasmatocyte and mainly distributed on the cell membrane.And the plasmatocyte also exist in HPOs.4.The source of silkworm plasmatocytes.Through the culture of HPO in vitro and remove the HPO in vivo,it shows that the plasmatocyte in circulatory hemolymph mainly come from HPO,rather than its proliferation.