Study On BmMLs And BmToll9 Immune Response Induced By LPS In Silkworm

TLRs(Toll-like-receptors),as immune receptors which play a critical role in immune are widely existed in insects and vertebrates.Recent years,there is a controversial issue about the evolutionary relationship of TLRs between the insects and vertebrates growing up.In addition to participating in the insect innate immunity,Toll is also involved in the control of the development about early Drosophila embryo dorsal-ventral axis.Regardless of any pathway,Toll in cells is to active sp?tzle ligand through external recognition factor and protease cascade.In mammals,TLRs,as pattern recognition receptors(pathogen recognition receptor,PRR),can directly identify the specific pathogen-associated molecular patterns(pathogen-associated molecular patterns,PAMPs).By analyzing the structure of TLR,we found that there are differences exist between vertebrates and insects,then what's function of the ancestors of TLR in the end? TLRs as key receptors in immune,existed before the differentiation of insects and vertebrates,or convergent evolution after differentiation,which became a controversial hot issue.Based on the above problems,silkworm as a model organism was studied with the following:(1)Analyze the relationship of TLR of mammals,insects etc in evolution by bioinformatic.(2)Regard silkworm BmToll9 as the target,by information analysis to study whether there are ML family proteins in silkworm genome,which can play a similar function with MD2,the ligand of TLR4 in mammals.(3)By ELISA to detect the binding properties between ML family proteins and different bacterial components.(4)By co-immunoprecipitation to detect whether silkworm BmToll9 capable of binding to the ligand ML family proteins.(5)Overexpressed Bm Toll9 in silkworm BmN cells to detect whether is capable of expression of a downstream antimicrobial peptides after LPS induction.We obtained the following results:1.By TLR phylogenetic tree analysis in mammals and insects,we found TLR of mammals and insects belong to two different branches,only Toll9 is an exception.The structure of receptors TLR4 and BmToll9 are very similar,both of them contain 745 amino acids,a typical transmembrane domain,and the amino acids 1-550 of the extracellular region are LRRs,as well as a c-flank cysteine motif.2.We found three ML protein sequence in silkworm genome database: BmPP,BmEsr16 and BmML-1 by BLAST and obtained BmPP,Bm Esr16 and Bm ML-1 sequences by RT-PCR,then amplified them as well as cloned them into the prokaryotic expression vector.Finally we transformed them into E.coli,purified to obtain BmPP,BmEsr16 and BmML-1 recombinant protein.By ELISA detectection,we drew a conclusion that BmEsr16 and BmML-1 with different bacterial components have binding characteristics,and speculated BmMLs may be involved in the identification of bacterial PAMP.3.Recombinant vector pIEX-4-BmToll9-exto(the extracellular region of BmToll9),pIEX-4-BmML-1,pIEX-4-BmPP,pIEX-4-BmEsr16 were constructed,then we transfected these vectors in S2 cells,and filtered stably expressing exogenous protein cell lines by BT antibiotic.4.We found BmPP and BmEsr16 can bind BmToll9 by co-immunoprecipitation,the binding implys the function of BmToll9 is similar to mammalian TLR4,which can be combined with MLs family proteins,finally to response to the innate immune pathway by LPS induction.5.BmToll9 gene was amplified and cloned to an expression vector pIEX-4-eGFP,then was thansfected in silkworm Bm12 cells,finally we observed BmToll9 successful expression by confocal microscopy and detected that antimicrobial peptide gene Moricin,Attacin transcriptional activity was significantly up-regulated after adding BmEsr16 and LPS induction by qRT-PCR.