首页|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

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

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  • 国家科技期刊平台
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马尾松毛虫质型多角体病毒（Dendrolimus punctatus cytoplasmic polyhedrosis virus，DpCPV）基因组 S5片段编码一个由881个氨基酸组成、分子量为101 kD 的非结构蛋白（NSP1）。为探寻 DpCPV NSP1蛋白的功能，将 DpCPV 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 kD）外，也检测到了80 kD 和20 kD 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 DpCPV 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。
Expression and Cellular Localization of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus NSP1 in Insects
Genome segment 5 of Dendrolimus puntatus cytoplasmic polyhedrosis virus(DpCPV)was predicted to encode a protein of 881 amino acids with a molecular mass of 101 kD non-structural protein(NSP1). In order to study the function of the DpCPV NSP1 protein, PCR primers were designed according to the S8 segment genome sequence. The antigen of DpCPV 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 to infect Autographa californica,midgut anatomical samples were taken in each day after infection,and the synthesis curve of NSP1 protein vs time was measured by Western blot. The results of Western blot indicated that the synthesized protein expressed by S5 segment was observed in the first day of infection ;in addition to full length protein(101 kD),20 kD and 80 kD protein bands also were detected,indicating that the expression of NSP1 was initiated in early stage and cleaving of NSP1 protein occurred. Moreover,for the first time,the DpCPV 1 S5 fragment was expressed in insect cells and subcellular localization of insects was observed. The results revealed that the NSP1 protein was present in the cytoplasm of the cells.

Dendrolimus puntatus cytoplasmic polyhedrosis virusNSP1polyclonal antibody preparationeukaryotic expressioncellular localization

靳亮、许翠萍、王金昌、关丽梅、张稳超、黄朝、高学梅、王洪秀

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江西省科学院微生物研究所，南昌 330029

江西省科学院鄱阳湖重点实验室，南昌 330029

鲁东大学生命科学学院，烟台 264025

江西省农业科学院农业应用微生物研究所，南昌 330200

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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

国家自然科学基金江西省科技重大专项基金中国科学院开放基金江西省科学院鄱阳湖中心重点实验室项目

312600312014ACF600022014AEM00319号-07

2016

10.13560/j.cnki.biotech.bull.1985.2016.11.024

生物技术通报
中国农业科学院农业信息研究所

生物技术通报

CSTPCDCSCD北大核心
影响因子：0.505
ISSN：1002-5464
年,卷(期)：2016.32(11)
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