马尾松毛虫质型多角体病毒NSP1蛋白在昆虫细胞表达及定位  

Expression and Cellular Localization of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus NSP1 in Insects

作　　者：靳亮[1,2] 许翠萍[3] 王金昌[1] 关丽梅[1] 张稳超 黄朝[1] 高学梅[1] 王洪秀[4] 

机构地区：[1]江西省科学院微生物研究所,南昌330029 [2]江西省科学院鄱阳湖重点实验室,南昌330029 [3]鲁东大学生命科学学院,烟台264025 [4]江西省农业科学院农业应用微生物研究所,南昌330200

出　　处：《生物技术通报》2016年第11期208-214,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31260031);江西省科技重大专项基金项目(2014ACF60002);中国科学院开放基金项目(2014AEM003);江西省科学院鄱阳湖中心重点实验室项目([2013]19号-07)

摘　　要：马尾松毛虫质型多角体病毒(Dendrolimus punctatus cytoplasmic polyhedrosis virus,Dp CPV)基因组S5片段编码一个由881个氨基酸组成、分子量为101 k D的非结构蛋白(NSP1)。为探寻Dp CPV NSP1蛋白的功能,将Dp CPV 1基因组S5-1片段的抗原区(1-600 bp)进行了原核表达,并将纯化蛋白免疫家兔,制备多克隆抗体。将稀释后的病毒液感染甜菜夜蛾幼虫,感染后每天解剖甜菜夜蛾并取中肠样品,通过Western blot检测NSP1蛋白的合成时间曲线。Western blot检测结果显示,S5片段表达的蛋白在病毒感染第1天就有合成,并且除了检测到全长的蛋白(101 k D)外,也检测到了80 k D和20 k D的蛋白条带,说明NSP1蛋白为早期表达蛋白,并且蛋白发生了切割。另外,首次对Dp CPV 1 S5片段进行了在昆虫细胞中表达和昆虫亚细胞定位,结果显示,昆虫细胞定位于细胞的细胞质。Genome segment 5 of Dendrolimus puntatus cytoplasmic polyhedrosis virus（Dp CPV）was predicted to encode a protein of881 amino acids with a molecular mass of 101 k D non-structural protein（NSP1）. In order to study the function of the Dp CPV NSP1 protein,PCR primers were designed according to the S8 segment genome sequence. The antigen of Dp CPV 1 genome S5-1 piece＇s area（1-600 bp）was in prokaryotic expression,and the polyclonal antibodies against the expressed proteins were raised in rabbits. The diluted virus was used toinfect Autographa californica,midgut anatomical samples were taken in each day after infection,and the synthesis curve of NSP1 protein vstime was measured by Western blot. The results of Western blot indicated that the synthesized protein expressed by S5 segment was observed inthe first day of infection;in addition to full length protein（101 k D）,20 k D and 80 k D protein bands also were detected,indicating that theexpression of NSP1 was initiated in early stage and cleaving of NSP1 protein occurred. Moreover,for the first time,the Dp CPV 1 S5 fragmentwas expressed in insect cells and subcellular localization of insects was observed. The results revealed that the NSP1 protein was present in thecytoplasm of the cells.

关 键 词：马尾松毛虫质型多角体病毒 NSP1 抗体制备 真核表达 细胞定位 

分 类 号：S763.306.4[农业科学—森林保护学]