Identification And Function Analysis Of Scavenger Receptor Class C BmSR-C In Silkworm,Bombyx Mori

The scavenger receptor as a multifunctional receptor belongs to the receptor supergene family,it is a type of transmembrane glycoprotein that exists on the cell surface.According to the different structure of amino acid,the scavenger receptor is divided into two subtypes:type I and type II.Both of them composed of N-terminal cytoplasmic domain,transmembrane domain,spacer domain,alpha helix coiled helix domain,collagen-like domain and C-terminal side Exotic composition.Goldstein and Brown's study of LDL accumulation in arteriosclerotic plaques patients with familial hypercholesterolemia showing low oxidized low-density lipoprotein(LDL)receptors revealed an enzyme which binds to acetylated low-density lipoprotein(AcLDL).In addition,it can bind to a variety of ligands such as polyanions and then transfer it to cells for degradation,even more participates in immune function.Therefore,it is named"Scavenger receptor".This receptor recognizes a wide range of ligands,including different gene products that bind modified LDLs and specific polyanionic ligands,but mainly recognize polyanions,including natural and modified low-density lipoproteins,apoptotic cells and pathogens,etc.As the research progressed,it has been found that the scavenger receptor can bind not only acetylated or oxidized LDL but also lipoteichoic acid(LTA),lipopolysaccharide(LPS),bacterial CpG DNA,and yeast?-glucan,multiple pathogen-associated molecular patterns(PAMPs),among others.As an important class of pattern recognition receptors(PRRs),scavenger receptors usually assist the elimination of pathogenic microorganisms by activating endocytosis,phagocytosis,or downstream signaling.The scavenger receptors that have been found are generally divided into eight types of A-H.Among them,the scavenger receptor A and the scavenger receptor B are studied more frequently and are closely related to atherosclerosis.They are currently more deeply involved in mammals research.The class C scavenger receptor(SR-C)is a type I transmembrane glycoprotein containing several distinct domains with the N-terminus located on the outer surface of the plasma membrane and the C-terminal region located in the cytoplasm.At present,C class scavenger receptor proteins have been identified in various species,such as Anopheles dirkonii,Aedesaegypti,Drosophila melanogaster,and Kuruuma shrimp.However,there have been less reports on their biological effects.DSR-CI in Drosophila melanogaster has phagocytosis that mediates gram positive and negative bacteria.The AaSRC of Aedesaegypti can recognize dengue virus and regulate the expression of antimicrobial peptides to eliminate the virus.The MjSRC of Kuruuma shrimp exerts an antiviral effect by enhancing the phagocytosis of white spot syndrome virus(WSSV)by blood cells in shrimp.In addition,MjSRC can also play an important role in the antibacterial immunity of Kuruuma shrimp by enhancing phagocytosis of blood cells and expression of antimicrobial peptides.Silkworm is not only an important economic insect,but also a Lepidoptera model insect.However,Bombyx mori class C scavenger receptors have not yet been cloned,and their related functions have not yet been reported.Studies on the C-class Scavenger receptor gene in silkworm will help to explore important physiological mechanisms such as lipid metabolism and immune response in Bombyx mori and Lepidoptera insects,enrich the study of silkworm gene function,and also provide other insects with special scales.The study of fin insects provides a theoretical basis.1.Cloning and analysis of BmSR-CAccording to the NCBI,silkworm genome database and bioinformatics related software,the primers were designed to obtain the B-type scavenger receptor gene BmSR-C of Bombyx mori and perform bioinformatics analysis.The full-length cDNA of BmSR-C is 2047 bp,of which the full-length ORF is 1821 bp and the 5'and 3'untranslated region(UTR)sequences are 118 bp and 32 bp in length,respectively.It is located on nscaf2800chromosome 27 and has a total length of 14456 bp that consists of 10 exons and 9 introns.The gene encodes 606 amino acids,and its predicted molecular weight is 67.54 kDa with an isoelectric point of 8.81.It has a typical C-type scavenger receptor structural feature,mainly by the CCP,MAN,and SO domains and a single transmembrane near the C-terminus domain composition.This was compared with multiple species of the C-class scavenger receptor homologous sequences of other species and phylogenetic analysis showed that BmSR-C is more conserved and related to a homologous protein of Spodopterafrugiperda,which is also a lepidopteran insect.2.The mRNA expression pattern of BmSR-CThe expression of BmSR-C in the silkworm larvae of the third instar was analyzed by qRT-PCR.The results showed that the expression of BmSR-C was highest in the third instar larvae of malpighian tubes,followed by blood cells and ovaries.There were no obvious expression in other organizations.Expression status of blood cells was also detected in each period of the silkworm,and found that there was a clear peak in the expression level in the blood cells during the silkworm's sleep and during the wandering stages.3.Preparation of Anti-BmSR-C polyclonal antibody and its specific detectionAccording to the specific sequence of BmSR-C,the prokaryotic expression recombinant vector was constructed and transformed into competent Rosseta strain.After it was induced by IPTG at different temperatures and different concentrations,the optimal induction conditions were found to be 37?at afinal concentration of 0.1 mM IPTG.The best conditions were selected for large-scale induction.High-purity recombinant proteins were purified by Ni~+affinity chromatography and the polyclonal antibodies were obtained after immunization of mice.The anti-serum titer of BmSR-C was determined by ELISA,and the titer was as high as 1:128000.Western blotting analysis showed that BmSR-C antibody specifically recognized the silkworm BmSR-C protein and its relative molecular weight was about 65 kDa,which was consistent with the predicted size.At the same time,western blotting was used to detect the expression of BmSR-C protein in silkworm tissues of the fifth instar and three days.The results showed that the expression level of BmSR-C protein was highest in blood cells of Bombyx mori,followed by malpighian tubule but not in other tissues.The immunofluorescence assay detected the expression of BmSR-C in the blood cells of silkworm.It was found that BmSR-C mainly expressed on the cell membrane,and can specifically label granulosa and plasmatocytes cells.4.Function study of BmSR-C in immuneIn vivo challenge experiments of silkworms with lipopolysaccharide(LPS),peptidoglycan(PGN),Pseudomonas aeruginosa,and Staphylococcus aureus showed that there was no significant change in the gene expression level of BmSR-C when challenged with LPS and Pseudomonas aeruginosa,as compared to the control group.When challenged with PGN,the expression level of BmSR-C significantly increased at 2 hours,peaked at 6 hours and then began to decline compared with the control group.Meanwhile,at 12 hours,the expression level remained significantly higher than the control group unlike when it was challenged with Staphylococcus aureus in which the change of BmSR-C expression level in the early stage was the same as that of the PGN until after 24 hours.However,there was no significant difference in the control group,which indicate that BmSR-C may be involved in the immune response of Gram-positive bacteria in the silkworm.The ability of direct binding of BmSR-C recombinant proteins to pathogenic bacteria was further tested by bacterial binding assay.Western blotting results showed that they could bind to Staphylococcus aureus and did not bind to Escherichia coli and Pseudomonas aeruginosa.According to the bacterial clearance experiment in the silkworm body,BmSR-C can significantly promote the removal of Staphylococcus aureus from the silkworm,and the promotion effect on the elimination of Escherichia coli and Pseudomonas aeruginosa is not obvious.