家蚕类胰岛素相关肽结合蛋白2（BmIBP2）参与BmCPV感染及其分子机制研究

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the most important viral pathogen to the silkworm. However, the mechanisms of silkworm against BmCPV have not been understood until now. In our previous study, it was found that the expression level of Bombyx mori insulin-related peptide binding protein 2 (BmIBP2) was significantly increased in the silkworm midgut after being infected by BmCPV; and the regulated expression multiples in the non-susceptible silkworm strain were higher than that in the susceptible silkworm strain. .In order to study the function of BmIBP2 involved in the response of silkworm to BmCPV, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to analyze expression charateristics of BmIBP2 in silkworm strains with different susceptibility after the BmCPV infection. Further, the co-immunoprecipitation techniques were also performed to screen and identify the interacting protein of BmIBP2, and to resolve its pathways and mechanisms in the response of silkworm to BmCPV. In addition, recombinant protein of BmIBP2 in vitro was expressed and RNA interference knockdown of BmIBP2 gene to determine its biological activities and the molecular mechanisms involved in the response of silkworm to BmCPV infection. .The results will not only help to understand the response mechanism of insect aganist the virus infection, but also provide a new clue to the biological activity of BmIBP2 and the molecular mechanisms of the silkworm against BmCPV infection.

家蚕质型多角体病毒（BmCPV）是引起家蚕病毒病的重要病原体之一，目前对家蚕抗BmCPV的防御机制还不是很清楚。我们前期研究发现，家蚕在感染BmCPV后中肠中的类胰岛素相关肽结合蛋白2（BmIBP2）的表达量明显上调，且在抗性品种中的上调表达水平远高于易感品种。为了进一步研究BmIBP2在家蚕抵御BmCPV感染过程中的分子机制，本项目拟采用荧光实时定量PCR技术，分析BmIBP2在不同抗性家蚕品种感染BmCPV后的时相表达特征；然后通过免疫共沉淀技术，筛选并鉴定BmIBP2的互作蛋白，解析其在家蚕抵御BmCPV感染过程中的作用途径及机理；最后，通过体外重组蛋白的表达和RNA干扰技术，明确该蛋白的生物学活性及其在家蚕抗BmCPV中的分子机制。研究成果将为深入了解昆虫抗病毒机制提供有力依据，并为进一步揭示BmIBP2的生物学功能及家蚕抵御BmCPV的分子机制奠定理论基础。

为了从分子水平上探索家蚕抵抗BmCPV感染的防御机制和不同家蚕品种对BmCPV的免疫调控机理，本研究通过高通量测序技术分析筛选了4008（BmCPV易感）和p50（BmCPV抗性）家蚕在感染BmCPV后的差异表达基因，发现家蚕中肠的类胰岛素相关肽结合蛋白2（Bombyx mori insulin-related peptide-binding protein, BmIBP2）基因的表达水平明显上调，且在抗性品种中的上调表达倍数高于易感品种。进一步研究发现，BmIBP2基因在其它几种病原侵染家蚕中肠中也有差异表达，说明该基因参与了家蚕对病原感染的应答与防御过程，并且具有重要的免疫防御功能；纯化的重组蛋白rBmIBP2对家蚕BmN细胞的增殖具有一定的抑制作用；BmIBP2基因被干扰沉默后，与对照组相比，BmIBP2基因呈现下调表达，家蚕BmN细胞增殖速度加快，同时检测到糖代谢相关呼吸酶基因呈现上调表达；同时，我们构建了转基因表达载体pIZT-IBP2对BmIBP2基因进行过表达，实验结果显示，与对照组相比家蚕BmN细胞增殖速度减慢，并且糖代谢呼吸酶相关基因呈现下调表达；.以EGFP为报告基因，利用Bac-to-Bac表达系统将BmIBP2和EGFP在家蚕细胞中进行融合表达，经同源重组获得vBmEGFP-IBP2重组杆状病毒穿梭质粒，转染家蚕细胞并用SDS-PAGE和Western-blot方法检测重组蛋白的表达，结果发现重组蛋白成功表达，大小约为55kDa。随后收集蛋白并利用EGFP作为抗原决定簇通过免疫共沉淀技术筛选与BmIBP2蛋白相互作用的宿主细胞蛋白。通过免疫共沉淀实验，我们筛选到一个与BmIBP2有相互作用的蛋白，经质谱鉴定为家蚕ATP合成酶（Bombyx mori ATP synthase）。经酵母双杂交实验验证，家蚕类胰岛素相关肽结合蛋白2与家蚕ATP合成酶之间的确存在相互作用。.上述研究结果不仅可为深入了解BmIBP2 参与家蚕抵抗BmCPV感染的途径及其作用机理提供了有力依据，而且为进一步揭示BmIBP2的生物学功能及家蚕抗BmCPV的分子机制奠定了良好的基础和依据。