Study On The Immune-related Functions Of Silkworm C-type Lectin S3

C-type lectin(CTL)is widely found in plants and animals,It has a conserved sugar recognition domain(CRD)structure and can recognize monosaccharide and polysaccharide molecules in the presence of calcium ions.The C-type lectin of insects is widely involved in mediating the immune response and the development process,especially the secretory CTL can play a role as the Pattern recognition receptor in mediating the humoral and cellular immune responses.In the past,most studies focused on humoral immunity mediated by C-type lectin,but few studies were conducted on how to mediate cellular immunity.In view of this,CTL-S3 mediated cellular immune response of silkworm was studied in this paper.First,the gene cloning,sequence analysis and phylogenetic analysis of C-type lectin S3(CTL-S3)were carried out.Then the tissue distribution and induced expression of the gene in bombyx mori were analyzed.The effects of recombinant proteins on bacterial binding and growth inhibition,as well as on bacterial clearance were studied.On this basis,the binding of CTL-S3 deletion mutants with bacteria,pathogen molecules and blood cells was analyzed.Finally,the interaction between the recombinant protein and Integrin ?5 subunit was analyzed.The results of this study laid a foundation for further study on the mechanism of CTL mediated cellular immune response and provided ideas for the prevention and control of silkworm disease and biological control of other pests.The main research results are as follows:1.Sequence analysis:The CTL-S3 open reading frame of silkworm was 672 bp,encoding 223 aa,with a 19 aa signal peptide and a 172 aa conservative structural domain CRD and belongs to the typical model S lecin(contains only one CRD of Simple type).MEGA 6.0 constructed homologous evolutionary tree and found that the homology of CTL-S3 was found in moths,butterflies,ants,beetles,flies,mosquitoes,bees,wasps,termites,aphids and beetles.The sequence homology with zebrafish and Shrimp is very low.2.Binding of recombinant protein to bacteria,cell wall components and blood cells:Western blot and ELISA experiments showed that CTL-S3 recombinant protein could bind to gram-positive and negative bacteria;can bind to blood cells and also bind to a variety of bacterial cell wall components.3.Tissue distribution and induced expression:qPCR showed that the expression level of CTL-S3 gene in the malpighian tubule was significantly down-regulated after the injection of Escherichia coli and Staphylococcus aureus.After Staphylococcus aureus injection,the expression level of CTL-S3 in the midgut was significantly down-regulated.After feeding for 6 h with Escherichia coli and Bacillus subtilis,the expression level of CTL-S3 gene in the whole worm was significantly up-regulated.In normal tissues,the expression of CTL-S3 gene is the highest in the malpighian tubule and the lowest in blood cells.4.Effects of C-type lectin on bacteria:According to the bacteriostasis and agglutination experiments,CTL-S3 recombinant protein can selectively inhibit the growth of Bacillus subtilis and cause the agglutination of Staphylococcus aureus.The CTL-S3 recombinant protein was found to promote the bacterial clearance in silkworm by antibody blocking and bacterial clearance experiments.5.Interaction between C-type lectin and integrin:Far-western analysis showed that there was an interaction between Flag-S3 fusion protein and integrin ?5 subunit.