家蚕抗BmNPV细胞因子的筛选和分析

doi:10.3864/j.issn.0578-1752.2018.04.018

摘要/Abstract

摘要： 【目的】探讨家蚕NF-kB类转录因子BmRelish在抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)感染的免疫应答中的作用，筛选和分析抗病毒细胞因子，完善对家蚕抗病毒免疫机制的认识。【方法】通过Western blot检测当BmNPV感染家蚕BmE细胞后BmRelish全长形式（BmRelish-FL）是否转变成其活性形式（BmRelish_act）；利用荧光定量PCR检测过表达BmRelish_act的细胞在感染BmNPV后胞内病毒DNA的相对水平，观测在被荧光标记的BmNPV（BmNPV-EGFP）感染后呈现EGFP荧光的细胞数，并与作为对照的未表达BmRelish_act的细胞相比较，以确定BmRelish是否参与抗病毒免疫；将新鲜细胞通过Transwell细胞共培养体系经过表达BmRelish_act的细胞上清培养液孵育后，定量检测在感染BmNPV后胞内病毒DNA的相对水平，以探讨过表达BmRelish_act的细胞是否产生并分泌抗病毒细胞因子；通过超滤等方法对过表达BmRelish_act的细胞上清培养液予以初步分离，将滤过液和截留液分别孵育新鲜细胞，定量检测感染BmNPV后胞内病毒DNA的相对水平，从而确定抗病毒细胞因子的分子量范围；用高温处理滤过液和截留液后再孵育新鲜细胞，定量检测感染BmNPV后胞内病毒DNA的相对水平以确定抗病毒细胞因子的属性；利用LC-MS/MS对滤过液中的多肽做进一步鉴定和分析。【结果】BmE细胞被BmNPV感染后，部分BmRelish-FL转变成其活性形式BmRelish_act；过表达BmRelish_act的细胞被BmNPV感染后，胞内病毒DNA的相对水平显著低于对照细胞，且呈现EGFP荧光的细胞数也较对照少；新鲜细胞经过表达BmRelish_act的细胞上清培养液孵育后，胞内病毒DNA的相对水平显著低于用对照细胞上清培养液孵育的细胞；用100和3 kD超滤管超滤过表达BmRelish_act的细胞上清培养液后，滤过液仍然具有显著降低所孵育细胞的胞内病毒DNA的相对水平，而截留液无此活性；经高温处理后，该细胞上清培养液不再具有降低所孵育细胞的胞内病毒DNA相对水平的作用；在滤过液中通过质谱鉴定到67个肽段，长度为9—45个氨基酸，富集到32个蛋白质，分子量均＞3 kD，其中9个蛋白质含有信号肽。【结论】BmNPV感染导致BmRelish的激活；活化的BmRelish促进细胞产生抗病毒细胞因子并分泌到上清培养液中；该细胞因子具有增强细胞抗病毒能力的活性，为分子量＜3 kD的多肽。

关键词: 家蚕, 家蚕核型多角体病毒, BmRelish, 细胞因子, 抗病毒免疫

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

王菲，李显扬，化晓婷，夏庆友. 家蚕抗BmNPV细胞因子的筛选和分析[J]. 中国农业科学, 2018, 51(4): 789-799.

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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