家蚕抗BmNPV细胞因子的筛选和分析

doi:10.3864/j.issn.0578-1752.2018.04.018

Abstract

Abstract: 【Objective】The objective of this study is to investigate the role of BmRelish, a NF-kB-like transcriptional factor in immune response against infection of Bombyx mori nucleopolyhedrovirus (BmNPV), and to screen and analyze the potential antiviral cytokines to better understand the antiviral immunity in silkworm (Bombyx mori).【Method】The generation of BmRelish active form (BmRelish_act) from full-length BmRelish (BmRelish-FL) in BmE cells after BmNPV infection was examined by Western blot, the relative level of viral DNA in BmRelish_act-expressing cells was evaluated by quantitative PCR and the amount of EGFP-positive cells was compared with control cells which did not express BmRelish_act after infection with EGFP-labeled BmNPV (BmNPV-EGFP) to investigate whether BmRelish participates in antiviral immunity. Naive cells were then incubated with supernatant medium of BmRelish_act-expressing cells in Transwell co-culture system followed by BmNPV infection, and the relative level of viral DNA in those cells was evaluated to determine whether antiviral cytokines were produced and secreted from BmRelish_act-expressing cells. Next, the supernatant medium of BmRelish_act-expressing cells was fractionated by ultrafiltration, and the filtrate and retenate fractions were incubated with naive cells respectively, the relative level of viral DNA in those cells was then evaluated to estimate the molecular mass of antiviral cytokines. This assay was also performed with heat-treated filtrate and retenate to determine the biological properties of antiviral cytokines. Finally, the supernatant medium of BmRelish_act-expressing cells was analyzed by LC-MS/MS.【Result】After BmNPV infection, BmRelish-FL was partially processed into its active form (BmRelish_act), and the relative level of viral DNA as well as the amount of BmNPV-EGFP positive cells in BmRelish_act-expressing cells was significantly lower than control cells. Incubation with supernatant medium of BmRelish_act-expressing cells also led to a remarkable decrease in the relative level of viral DNA in naive cells after BmNPV infection. Incubation with filtrate fractions from the supernatant medium of BmRelish_act-expressing cells fractionated by centrifugal filter with cut-off size of 100 and 3 kD maintained the antiviral activity, while retenate fractions did not. The antiviral activity can be removed by heating. A total of 67 peptides consisting of 9-45 amino acids and derived from 32 proteins were identified by LC-MS/MS from the filtrate fraction of supernatant medium. Those proteins all have molecular mass＞3 kD and 9 of them contain signal peptide.【Conclusion】BmRelish is activated in response to BmNPV infection and participates in antiviral immunity by promoting the production of antiviral cytokines, which are secreted into the supernatant medium and enhance the anti-BmNPV immunity of naive cells. Those anti-BmNPV cytokines are small peptides with molecular mass＜3 kD cleaved from larger proteins.

Key words: silkworm (Bombyx mori), BmNPV, BmRelish, cytokine, antiviral immunity

WANG Fei, LI XianYang, HUA XiaoTing, XIA QingYou. Screening and Analysis of Anti-BmNPV Cytokines in Silkworm (Bombyx mori)[J].Scientia Agricultura Sinica, 2018, 51(4): 789-799.

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Screening and Analysis of Anti-BmNPV Cytokines in Silkworm (Bombyx mori)

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