Differential Expression And Its Functional Analysis Of Immune System Related Genes In BmNPV-infected Silkworm

Proteomics is an important means about studying the endocrine metabolism and the function of proteins and related genes.Through this technology,we understand the host immune system regulation,metabolic pathway regulation and other series of organism stress response.When the host is infected by an external pathogenic microorganism,humoral immunity and cellular immunity are activated,mainly due to changes in the expression levels of related proteins involved in immune regulation.Therefore,it is very important to study the regulation of immune-related proteins to explore the the interactions between the microbial pathogenesis and host immune system after the host infected with the pathogen.Some of these related proteins involve in the allergic inflammatory response of large mammals,while others are beneficial means for the host effectively prevent pathogenic microorganisms.But very few proteins can also participate in the pathogens infection and increase the damage of pathogens.In this experiment,three different proteomics techniques were used to study the regulation of immune-related proteins and genes in BmNPV-infected silkworm.1.Optimization and establishment of Two dimensional electrophoresis in silkworm pupaThe experiment utilize the universal silkworm pupa as the material.Diferent purification and extraction methods,three types of the focusing times and two kinds of the coomassie blue stainings to influence the 2-DE maps.Results indicated that the most result is through TCA-acetone to overnight-settlement extraction,isoelectric focusing with 8000 v,80000vh is the best.pH4-7 IPG strip and equilibration time is the 15 mins could remove vertical sripes to get the better optimization results.Dyeing methods have their advantages and disadvantages.This experiment lays foundation for the research of proteomics of silkworm pupa.2.Baculoviral infection reduces expression of four allergen proteins of silkworm pupaSeveral proteins expressed in silkworm pupae cause severe allergic reactions in humans and animals.Interestingly,some baculovirus vectors have been shown to alter host gene and its expression in insect cells,but this has not been confirmed in silkworm.Here,we analyzed the effects of infection with an empty B.mori baculovirus(BmNPV)vector on silkworm pupal protein expression.Using a proteomics approach,the allergens,thiol peroxiredoxin(Jafrac1),27-kDa glycoprotein(p27k),arginine kinase,and paramyosin as well as 32 additional differentially expressed proteins,were identified.Down-regulation of the mRNA expression of the four known allergens was observed after BmNPV infection;subsequent changes in protein expression were confirmed by western blotting using polyclonal antibodies prepared with recombinant proteins of four allergens.Collectively,these data indicate that the four known allergens of silkworm pupae can be reduced by infection with an empty BmNPV to increase the safety of silkworm pupa-based exogenous protein expression and drug delivery of oral pharmaceuticals.In addition,the four recombinant allergen proteins contribute to the diagnosis of allergic diseases of silkworm pupa.3.Effects of Bombyx mori nuclear polyhedrosis virus on serpin and antibacterial peptide expression in B.morisilkworm proteins were screened using proteomics after Bombyx mori nuclear polyhedrosis virus(BmNPV)infection,and 6 antimicrobial peptides and 12 serpins were identified.The mRNA expression levels of these 18 proteins were examined at different times.The results indicated that attacin had the highest expression level,while serpin-5 and cecropin-D exhibited a negative regulatory correlation.These results provide a significant step toward a deeper understanding of B.mori immunoregulation.4.iTRAQ proteomic analysis of BmNPV-silkworm interactionsProteomes were analyzed before and after infection using isobaric tags for relative and absolute quantitation and LC-MS.A total of 456 differentially expressed proteins were identified,of which 179 were upregulated and 193 were downregulated.Changes in expression were validated by western blot for several proteins.Eleven of the differentially expressed proteins were involved in immunity.For example,modular serine protease and cecropin,which were downregulated,facilitate Toll and Imd signaling,while autophagy-related protein 3,which was upregulated,protects cells against oxidative damage.Collectively,the data highlight the unique interactions of baculovirus with the silkworm immune system.