Cloning And Expression Analysis Of ApCTL Gene From Antheraea Pernyi

As invertebrates,Antheraea pernyi lack title adaptive immune system,and it mainly depends on the developed innate immune system to resist the invasion of pathogenic microorganisms.The natural immune system of insects mainly includes three parts:physical defense,humoral immunity and cellular immunity.Innate immunity begins with the identification of pathogenic microorganisms.This process is achieved by pattern recognition receptors?PRPs?in insects recognize and bind pathogen-specific molecular patterns?pathogen-associated molecular patterns,PAMPs?.The interaction of pattern recognition receptors with pathogen-associated molecular patterns induces a series of immune responses,including phagocytosis,melanization and the production of antimicrobial peptides,antiviral factors,etc,activating the host defense system.Because of its ability to bind carbohydrates,lectins play an important role in many biological processes.Including protein synthesis and transportation,cell signal transduction,pathogenic microorganism identification,etc.As a pattern recognition receptor,lectin contains a specific carbohydrate recognition domain CRD.Different CRDs have unique structures and can identify specific molecular molecules on the surface of pathogenic microorganisms,thus activating the insect immune defense system.Among them,C-type lectin is a major family of innate immune pattern recognition receptors,and also one of the largest animal lectin families.It has good binding ability to glycoproteins and glycolipids on the surface of pathogens.In this study,A.pernyi pupae were injected with 5 different microorganisms,and quantitative RT-PCR was performed,to explore the differences in expression of lectin genes in different tissues under different inducing conditions,and to provide references for further studies on the mechanism and activation pathway of lectin in the immune system of the Antheraea pernyi.The result suggest that the different lectins may have different functions in different tissues of A.pernyi.Bioinformatics results showed that the ApCTL gene was 912 bp long and encoded 304 amino acids.The amino acids 1-16 were signal peptides.The sequence contained two conserved carbohydrate recognition domains,each domain contained a specific binding tripeptide.ApCTL was cloned into a transfer vector of Antheraea pernyi nucleopolyhedrovirus?ApNPV?pApM748BE.The recombinant plasmid DNA was obtained and used to cotransfect cultured Tn-Hi5 cells together with ApNPV DNA.The recombinant virus was selected with limited dilution method.A.pernyi Pupae were infected by this recombinant virus and the expression of target gene was analyzed using SDS-PAGE and Western blotting methods.The result indicates that the agglutination activity of the ApCTL against the bacteria was Ca²⁺dependent,and ApCTL also have good agglutination activity with the rabbit red blood cells.