Cloning,Expression And Functional Study Of C-type Lectin IML-3 From Bombyx Mori

Insect C-type lectin（CTL）is a kind of pattern recognition receptor family that can recognize glycosylated structure of pathogen surface rely on Ca²⁺,which is very important to initiate innate immune response and resist infection by pathogenic microorganisms.C-type lectins recognize specific glycosyl through the Carbohydrate recognition domain（CRD）.C-type lectins with two CRDs,often called Immulectin,are a characteristic structural feature of lepidoptera and widely involved in the regulation of humoral and cellular immune responses.BmIML-3 is a C-type lectin with two CRDs in Bombyx mori,and its immune function has not been reported so far.This study focused on the immune function of BmIML-3:Bioinformatics analysis of BmIML-3 gene;Cloning of BmIML-3 gene,prokaryotic expression and purification of recombinant protein BmIML-3,CRD-1 and CRD-2;The expression patterns of BmIML-3 gene in different development stages and tissues of Bombyx mori,and the expression patterns induced by fungi and bacteria;Binding of recombinant proteins to bacteria,pathogen molecular patterns,and hemocyte;The agglutination of recombinant proteins to pathogenic microorganisms;Antibacterial activity of recombinant protein;Recombinant proteins mediated phagocytosis and encapsulation reaction of Bombyx mori hemocyte;Effect of recombinant protein on hemocyte agglutination.The main research results are as follows:1.Bioinformatics analysis results:The total length of BmIML-3 gene was 6424 bp,theCDS region was 1174 bp,and the ORF region was 939 bp.BmIML-3 consists of 312 amino acids with a theoretical size of 35.14 k Da and contains two CRD domains.The prokaryotic expression of BmIML-3,CRD-1 and CRD-2 recombinant protein showed that the molecular weight of r BmIML-3 was 35 k Da,and the molecular weight of CRD-1 and CRD-2 were 14k Da and 16 k Da,respectively,which was consistent with the predicted results.2.Expression distribution and induced expression rule in tissues and development stage:The results of fluorescence quantitative analysis showed that BmIML-3 gene was mainly expressed in the late third instar larvae,the fifth instar larvae and adults.The expression pattern of tissue distribution showed that BmIML-3 gene was mainly expressed in the fat bodies and martensite canal of the 5th instar larvae.Both Escherichia coli and Staphylococcus aureus could induce a significant increase in BmIML-3 gene expression.BmIML-3 gene expression was significantly up-regulated in hemocyte at 12 h and 48 h after infection,and in epidermis at 48 h.The conidial induction results of Beauveria bassiana showed that BmIML-3 gene was significantly up-regulated in hemocyte,fat bodies and epidermis at 12 h after injection induction,and significantly up-regulated 6 times in epidermis at 48 h after injection induction.3.Studies on the immune function of recombinant proteins BmIML-3,CRD-1 and CRD-2 in vitro:Microbial binding experiments showed that all recombinant proteins could bind to Escherichia coli and Staphylococcus aureus,but the binding ability of BmIML-3 was stronger than that of CRD-1 and CRD-2.BmIML-3,CRD-1 and CRD-2 can bind to Laminarin,Chitin,Curdlan,Zymosan and lipopolysaccharide（LPS）,and can also bind to hemocyte.BmIML-3 and CRD-2 can bind to peptidoglycan（PGN-EK and PGN-SA）.The agglutination experiment showed that the recombinant proteins BmIML-3 and CRD-2 had enhanced the agglutination ability of Escherichia coli,Bacillus subtilis and Staphylococcus aureus mediated by Ca²⁺,while CRD-1 had no significant effect on the agglutination effect of staphylococcus aureus.The presence of EDTA and sugar molecules could inhibit the agglutination effect of proteins on bacteria.The results showed that the recombinant proteins BmIML-3,CRD-1 and CRD-2 could inhibit the growth activity of Staphylococcus aureus,Serratia marcescens and Bacillus subtilis.The recombinant proteins BmIML-3 and CRD-2significantly enhance hemocytosis and cytophagy,and promote hemagglutination,mediated by Ca²⁺.In conclusion,this study suggests that BmIML-3 has multiple functions in the innate immune response of Bombyx mori.These results laid a foundation for further study on the role of BmIML-3 in the immune defense system of Bombyx mori and provided a new idea for the control of Bombyx mori diseases.