Gene Hsc70-4 promotes the replication and proliferation of Bombyx mori nucleopolyhedrovirus
Gene Hsc70-4 promotes the replication and proliferation of Bombyx mori nucleopolyhedrovirus
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DOI：
10.13343/j.cnki.wsxb.20180377
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发表时间：
2019-01-01
期刊：
影响因子：
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通讯作者：
Lu, Cheng
中科院分区：
文献类型：
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作者：
Chen, Peng;Bao, Xiyan;Lu, Cheng
[目的]热休克反应是机体对环境应激的一种重要的细胞防御策略。鉴定热休克蛋白在杆状病毒感染过程中的功能并揭示其作用的分子机制将为理解宿主与病毒相互作用的分子基础提供理论基础。[方法]采用分子克隆的方法克隆Bmhsc70-4基因，用BioEDIT和GeneDoc进行多序列比对。Bmhsc70-4基因在真核细胞中高效表达，经CRISPR/Cas9基因编辑系统敲除。用实时定量聚合酶链式反应检测相应基因的表达。通过检测Caspase-9和Caspase-3/7的活性来检测Bmhsc70-4基因对细胞凋亡的影响。免疫荧光法证实BmHSC70-4与BmIAP的共定位，免疫沉淀法进一步证实BmHSC70-4与BmIAP的相互作用。[结果]Bmhsc70-4基因的开放阅读框为1950bp，编码649个氨基酸，在昆虫中高度保守。在BmNPV感染过程中，Bmhsc70-4基因表达上调。Bmhsc70-4基因的过表达可促进BmNPV的增殖，而Bmhsc70-4基因的敲除抑制了BmNPV的增殖，表明Bmhsc70-4基因的表达有利于BmNPV的增殖。我们证实了Bmhsc70-4基因可以抑制家蚕细胞的凋亡。免疫荧光分析表明，BmHSC70-4和BmIAP共定位于细胞质中，免疫共沉淀表明它们可以相互作用。Bmhsc70-4基因可促进BmNPV感染过程中Bmiap基因的表达。[结论]Bmhsc70-4基因具有抑制家蚕细胞凋亡的作用，在BmNPV感染家蚕细胞的过程中与BmIAP相互作用，促进BmNPV的复制和增殖。
[Objective] Heat shock response is an important cellular defense strategy to environmental stresses. Identification of the function of heat shock protein in the process of baculovirus infection and revealing the molecular mechanism of its role will provide a theoretical basis for understanding the molecular basis of host-virus interaction. [Methods] Bmhsc70-4 gene was cloned by molecular cloning, and multiple sequence alignment was performed with BioEdit and GeneDoc. The Bmhsc70-4 gene was overexpressed by eukaryotic expression and knocked out by CRISPR/Cas9 gene editing system. The expression of the corresponding genes was detected by quantitative real-time PCR. The effect of Bmhsc70-4 gene on apoptosis was determined by detecting the activity of Caspase-9 and Caspase-3/7. The co-localization of BmHSC70-4 and BmIAP was verified by immunofluorescence, and their interaction was further verified by immunoprecipitation. [Results] The open reading frame of Bmhsc70-4 gene was 1950 bp, encoding 649 amino acids and it was highly conserved among insects. Bmhsc70-4 gene was up-regulated during BmNPV infection. Overexpression of Bmhsc70-4 gene could promote the proliferation of BmNPV, and knockout of Bmhsc70-4 gene inhibited the proliferation of BmNPV, indicating that the expression of Bmhsc70-4 gene was conducive to the proliferation of BmNPV. We confirmed that Bmhsc70-4 gene can inhibit the apoptosis of silkworm cells. Immunofluorescence assay showed that BmHSC70-4 and BmIAP were co-localized in the cytoplasm and immunocoprecipitation showed that they could interact with each other. Bmhsc70-4 gene could promote the expression of Bmiap gene during BmNPV infection. [Conclusion] Bmhsc70-4 gene had the function of inhibiting apoptosis of silkworm cells, and it could interact with BmIAP and promote BmNPV replication and proliferation during the process of BmNPV infecting silkworm cells.