Differential Proteomic Analysis And Differentially Expressed Genes Of Immune-challenged Musca Domestica Larvae

The house flies(Musca domestica)live in surroundings full of various microbes. They can spread many diseases owing to a lot of pathogen on their body surface. House flies have been implicated in the spread of over 100 pathogens that may cause diseases in humans and animals,including typhoid,cholera,bacillary dysentery, tuberculosis,anthrax ophthalmia and infantile diarrhea,as well as parasitic worms. But they can thrive without causing infection.Therefore it is presumed that the house flies have unique immune defense mechanism.House flies lack adaptive immune system like the vertebrate.But they can produce immune active materials quickly when infected by bacteria and other pathogens.The active materials contain some antimicrobial peptides and lysozymes etc.They can kill and inhibit not only various viruses,bacteria,fungi and parasites but also some cancer cells.Most of them are safe to humans.The anticancer effect of the active materials from flies are the focus of studies.Some antibacterial peptides and other active materials have been separated from Drosophilas,Boettcherisca peregrinas and Anopheles gambiae.Therefore,studies in the immune system of the house flies have important academic value and good application prospect.In this study,comparative proteomic analysis was performed to detect the change of the protein expression in hemolymph of bacterial challenged and unchallenged house fly larvae.We also identified several novel immune response proteins in addition to those already reported immune response proteins in insects.And a suppression subtractive hybridization cDNA library was constracted.Differentially expressed genes were identified in larvae of house fly challenged for 12 hours with Escherichia coli and Staphylococcus aureus versus those from unchallenged house fly. A superoxide dismutase gene was cloned from the house flies by homology cloning. The transcriptional pattern of Cu/Zn SOD gene in house fly chanllenged with bacteria for different periods was analyzed using RT-PCR.The antibody was obtained with purified recombinant Cu/Zn SOD,and then the translational pattern of this gene was characterized using Western blot.Furthermore,the expression patterns of the gene encoding ubiquitin were studied.And the recombinant protein was purified for further analysis of its characteristic.The results of the studies are as follows:1.To understand immune response of the house fly(Musca domestica)to bacterial infection at the protein level,we performed comparative proteomic analysis. The protein expressional profiles in hemolymph of bacterial challenged and unchallenged larvae were examined by two-dimensional gel electrophoresis(2-DE). Among～350 reproducibly detected protein spots on each gel,288 present at uniform levels in all samples,11 were up-regulated,37 were down-regulated in hemolymph from immune-challenged larvae,and 14 spots detected exclusively in challenged larvae.Mass spectrometry identified 35 differentially expressed proteins,including immune related proteins,such as Gram-negative bacteria-binding protein 1, Peptidoglycan-recognition protein- LF,Nuclear factor NF-κB p110 subunit and Lysozyme X.Proteins involved in detoxification and proteins with a potential role in the immune response were also identified,such as superoxide dismutase,glutathione S-transferase,cytochrome P450.We also identified several novel immune response proteins including DCN1-like protein and cAMP-dependent protein kinase,in addition to those already reported immune response proteins in insects.The results suggest that these proteins may be involved in innate immunity,providing new insights for the innate immune mechanisms of the house fly.Semi-quantitative reverse-transcription polymerase chain reaction(RT-PCR)analysis confirmed the changes of Lysozyme X,Glutathione S-transferase D5 and Superoxide dismutase [Cu-Zn]at mRNA levels,indicating that these proteins were also regulated at transcriptional levels.2.A suppression subtractive hybridization cDNA library of Musca domestica was constructed for identifing differentially expressing genes in larva of house fly challenged for 12 hours with Escherichia coli and Staphylococcus aureus versus those from unchallenged house fly.First,total RNAs were isolated from these different house flies.Then,mRNAs were purified from these total RNAs.After that, Single-strand cDNAs and double-strand cDNAs were synthesized.After digestion with restriction enzyme of Rsa,cDNAs were obtained,cDNAs from challenged house fly,named tester,then were divided into two groups and ligated to the specific adaptor 1 and adaptor 2R respectively.The cDNAs from unchallenged house fly, named driver,didn't ligated the adaptors.The cDNAs from tester and driver hybridized twice,then underwent two times of nested PCR,subtractive cDNAs with adaptor 1 and adaptor 2 in tester were obtained and enriched.The subtractive cDNAs were used to constructed the library with pGEM-T Easy vector and transfored into DH5α,the subtractive library of Musca domestica was set up and amplificated. Screening positive clones by PCR shows that they all contain recombined plasmids with 200bp-750bp inserts.After sequence alignment by BLASTx,several ESTs of immunity related genes and oxidoreductase protein genes were found,such as defensin,cytochrome oxidase subunitⅡ,lysozyme,serine protease,catalase, oxidoreductase.Thus,the cDNA subtractive library of Musca domestica would provide sold basis for screening immunity related genes of the house fly.3.The full length cDNA of the Cu/Zn superoxide dismutase(Cu/Zn SOD)was cloned from house fly.The full-length Cu/Zn SOD cDNA(734bp)of Musca domestica was included a 9 bp 5' untranslated region(UTR),a 462 bp open reading frame and a 263 bp untranslated region in the 3' UTR with a 17 bp poly A tail.The ORF encoded a 153 amino acid protein with no signal peptide.It has a calculated molecular mass of 15.6 kDa and a predicted isoelectric point of 5.67.The mature protein included one Sod_Cu domain.To further confirm the expression pattems of the gene encoding Cu/Zn SOD, RT-PCR was used to study the differences in gene expression after bacterial challenged in different time.The result demonstrated that the expression of Cu/Zn SOD increase from 3h after bacterial challenged,reached to a maximum value at 6h and was decreased post 12h challenged.This result was consistent with the result derived from comparative proteomic analysis of the house fly larvae to bacterial infection.This result suggested that the Cu/Zn SOD protein may have the function in the innate immunity of the house fly.The sequence coding for Cu/Zn SOD protein was amplified and ligated into expression vector pGEX-4T-1.The recombinant vector was transformed into competent E.coli BL21(DE3)host cells.The recombinant Cu/Zn SOD was epressed in the supernatant and was purified with Glutathione Sepharose 4B chromatography. Polyclonal antiserum was obtained from rabbit after continuous injection of recombinant Cu/Zn SOD.With polyclonal antibodies we also studied the expression patterns of the nature protein in different tissues including hemolymph,midgut and fatbody after bacterial challenged.After SDS-PAGE,different proteins were transfered to the Nitrocellulose membrane.After incubation with polyclonal antibodies and second antibody (HRP-conjugated goat anti-rabbit IgG)and visualization the natural Cu/Zn SOD was characterized as a 16kDa protein in hemolymph,midgut and fatbody,After bacterial challenged,the expressions in hemolymph,midgut and fatbody were all increased,but was increased weakly in hemolymph.The activity of recombinant Cu/Zn SOD was detected with Native-PAGE(10%), and the result indicated that the specific band of Cu/Zn SOD with high activity was found after the induction with IPTG for 3h.Furthermore,purified Cu/Zn SOD showed high activity.Besides,weak activity was also found before induction,which suggested that a small amount of leakage expression of Cu/Zn SOD maybe take place in the transformed strain.4.The expression pattern of Mub_S27in the house fly challenged with bacteria was analyzed by RT-PCR.The result showed that after Staphylococcus aureus and E.coli infection,the expression of ubiquitin-fusion gene was upregulated from 2 to 8 h and then returned to the original level.We have also identified the NF-κB protein in the house fly larvae by proteomic analysis.It was inducing expression after bacterial challenge.Hence,we can speculate that the up-regulated Ub could cause TRAF6 ubiquitination and eventually activate the transcription factor NF-κB.The activated NF-κB would then induce the expression of a number of antimicrobial peptides. Therefore Mub_S27maybe involved in the defense against bacteria.The sequence coding for mature protein was amplified and ligated into expression vector pET-30a(+).The recombinant vector was transformed into competent E.coli BL21(DE3)host cells.The recombinant Mub_S27was in the supernatant and was purified with His Bind resin chromatography.This study will enhance the understanding of the immune system in the house fly; enrich the knowledge of the innate immunity in insect.