He Lei, Hu Xiaolong, Zhu Min, Liang Zi, Chen Fei, Zhu Liyuan, Kuang Sulan, Cao Guangli, Xue Renyu, Gong Chengliang
School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, China.
School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, China; National Engineering Laboratory for Modern Silk, Soochow University, Suzhou 215123, China.
Gene. 2017 Sep 5;627:343-350. doi: 10.1016/j.gene.2017.06.048. Epub 2017 Jun 28.
The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1-S10). The segment 7 (S7) encodes 50kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7.
家蚕细胞质型多角体病毒(BmCPV)的基因组包含10条双链RNA片段(S1 - S10)。片段7(S7)编码50kDa的蛋白质,该蛋白质被认为是一种结构蛋白。p50在病毒生命周期中的表达模式和功能仍不清楚。在本研究中,用免疫小鼠制备了病毒结构蛋白7(VP7)多克隆抗体,以探究家蚕细胞质型多角体病毒中小VP7基因编码蛋白的存在情况。通过在BmN细胞中过表达来研究vp7基因的表达模式。除了VP7外,用蛋白质免疫印迹技术鉴定出了一条补充条带。还用蛋白质免疫印迹技术检测了病毒粒子、BmCPV感染的细胞和中肠。在相应样品中分别检测到4条、2条和5条条带。通过RNA干扰降低vp7基因的表达水平,可使BmCPV基因组在家蚕培养细胞和中肠中的复制减少。在免疫沉淀实验中,使用针对VP7的多克隆抗血清,在BmCPV感染的中肠中检测到一条额外的较短条带,然后用质谱(MS)对该条带进行分析,MS结果表明从家蚕中鉴定出一种候选相互作用蛋白(VP7电压依赖性阴离子选择性通道样异构体,VDAC)。我们得出结论,新的病毒产物是通过渗漏扫描机制产生的,并且VDAC可能是与VP7相互作用的蛋白。