hsc70-4基因促进家蚕核型多角体病毒复制增殖

Gene Hsc70-4 promotes the replication and proliferation of Bombyx mori nucleopolyhedrovirus

摘要:

[目的]热休克应答（heat shock response，HSR）是机体细胞应对环境压力的一种重要防御策略，鉴定热休克蛋白在杆状病毒侵染宿主过程中的功能，并揭示其作用的分子机制，为探明宿主与病毒相互作用的分子基础提供理论依据。[方法]通过分子克隆技术对Bmhsc70-4基因进行克隆，并利用BioEdit及GeneDoc对其进行多序列比对分析；分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对Bmhsc70-4基因进行过表达和敲除；利用荧光定量PCR技术检测相应基因的表达量；通过对Caspase-9和Caspase-3/7活性的检测确定Bmhsc70-4基因对细胞凋亡的影响；通过免疫荧光验证BmHSC70-4和BmIAP的共定位情况，并进一步通过免疫共沉淀验证它们的相互作用。[结果]Bmhsc70-4基因开放阅读框为1950 bp，编码649个氨基酸，在昆虫间具有较高的保守性；BmNPV能够诱导Bmhsc70-4基因上调表达，过表达Bmhsc70-4基因能够促进BmNPV的增殖，敲除Bmhsc70-4基因能够抑制BmNPV的增殖，表明Bmhsc70-4基因的表达利于BmNPV的增殖；Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能；荧光共定位显示BmHSC70-4和BmIAP共定位于细胞质中，免疫共沉淀结果表明两者可以相互作用；BmNPV侵染过程中Bmhsc70-4基因能够促进Bmiap基因的表达。[结论]Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能，在BmNPV侵染家蚕细胞过程中，能够与BmIAP相互作用，并促进BmNPV复制增殖。

Abstract:

[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.