Immune Activity Of Bombyx Mori Thymosin And Its Expressions Regulation In The Process Of Bombyx Mori Infection With BmNPV

Thymosins distribute in multiple species, highly conserve during evolution, and play a crucial role in the immune and prevention of disease. However, we know little about the physiological functions of insect thymosins. In a previous study,we identified a gene from a c DNA library of silkworm pupae, which has an open reading frame(ORF) of 399 bp encoding 132 amino acids and contains two THY(Thymosin) conservative domain.Therefore, the gene is named as Bm THY. In this study, we analyzed the structure and chemical properties of Bm THY.This study focused on immune activity of Bombyx mori thymosin and its regulation of expression in process of Bombyx mori infection with Bm NPV.Bioinformatics analysis showed that the isoelectric point of Bm THY is 4.83, the molecular weight of 14.74 k Da and there is no hydrophobic group exist which illustrate that Bm THY has strong hydrophilicity. Bm THY is a kind of acidic protein which does not exist signal peptide and phosphorylation site. There is a glycosylation site which is thought to play a crucial role in cell metabolism.Transcription and expression level of Bm THY in Bm N cells and fifth instar larvae are down-regulated after Bombyx mori was infected with Bm NPV. Bm THY transcription and expression level of Bm N cells is 30.89%, 35.88%, 20.63% and 65.50%、66.46%, 62.51% to control group after Bombyx mori was infected with Bm NPV at 48 h, 72 h, 96 h respectively. While Bm THY transcription and expression level of fifth instar larvae is 24.51%, 63.53%, 38.76% and 86.25%, 57.76%, 69.70% to control group after silkworms were infected with Bm NPV at 48 h, 72 h, 96 h respectively.After investigating Bm THY transcriptional changes in different tissues after silkworms challenged with Bm NPV, we found that there were obvious differences among different tissues. Compared with the five instars control group which did not infected with Bm NPV, Bm THY m RNA from malpighian tube at the time point of 12 h, 24 h and 48 h after infectionwere 46.91%, 18.19%, 29.50% of that in control respectively, while Bm THY m RNA from midgut at 24 h, 48 h, 96 h after infection were 56.96%, 76.28% and 38.22% of that before infection. Bm THY m RNA in Both malpighian and midgut showed a down-regulation trend after silkworms infected by Bm NPV. However transcription level of Bm THY in spiracle, hemolymph and fat body showed a up and down fluctuation after Bm NPV infection.To determine the antiviral effects of r Bm THY against Bm NPV infection, we prepared r Bm THY protein contain His tag expressed in E.coli expression system. Cell viability of Bm N cells which infected with Bm NPV was 35.2±4.2%. However, the cell viability of Bm NPV-infected cells treated with r Bm THY at different concentrations( 0.04, 0.4, 4 μg/m L) were 69 ± 0.9%, 74.7 ± 3.1%, 84.9 ± 3.7% at 48 h of incubation, respectively, and 40.2 ± 2.8%, 46.2 ± 5.9%, 84.9 ± 2.7% at 72 h, respectively. Cells infected with Bm NPV showed considerable morphological alterations including deformation and shrinkage. However, cell morphologies, such as shape intactness, light transmission, and surface smoothness, in the drug treatment group were better than that of the viral control group.The cocooning rate of Bm NPV-licked fifth instar larvae treated with different concentrations of r Bm THY( 340, 34, 3.4 μg/m L) were 73.6%、66.2%、59.4% at 9 day of incubation, respectively, which significantly higher than the control group(45.4%). The cocooning rate of fifth instar larvae with subcutaneous inoculation of Bm NPV and treatment with different concentrations of r Bm THY( 340, 34, 3.4 μg/m L) were 56.2%、46%、42.4%、37.8%. The results showed r Bm THY protein can improve the cocooning rate of fifth instar larvae after Bm NPV infection indicating its antiviral effect.These findings suggest that Bm THY is a resistance gene against Bm NPV. Bm THY exerts immunomodulatory effects on Bombyx mori. Recombinant Bm THY has an antiviral effect for Bombyx mori against BmNPV infection.