马尾松毛虫质型多角体病毒结构蛋白的抗体制备及免疫性

Polyclonal antibody preparation and immune activity of structural proteins VP1, VP3 and Vp5 of Dendrolimus puntatus cytoplasmic polyhedrosis virus

质型多角体病毒的病毒粒子结构蛋白是由衣壳蛋白VP1(Capsid protein,CP)、塔状蛋白VP3(Turret protein,TP)和大突起蛋白VP5(Large protrusion protein,LPP)组成,这3个蛋白分别由病毒基因组片段S1、S4和S7编码.为了解质型多角体病毒结构蛋白的免疫定位情况,本研究从马尾松毛虫质型多角体病毒湖南株中提取病毒核酸,回收、纯化S1、S4和S7片段,经RT-PCR扩增得到对应片段的ORF全长或抗原区的片段,克隆于原核表达载体上,在大肠杆菌BL21菌株中进行了表达,免疫家兔制备了多克隆抗体.Western blot结果显示DpCPV基因组的S1、S4和S7片段分别编码其结构蛋白VP1、VP3和VP5,与推测的结果一致.免疫胶体金的方法也证明S1、S4和S7编码的蛋白定位于病毒粒子衣壳的外表面.本研究首次通过免疫学方法确定了质型多角体病毒结构蛋白在病毒粒子上的定位,为质型多角体病毒的结构蛋白功能和侵染机制研究提供了免疫学方面的基础,对质型多角体病毒的研究有较重要的意义.

Virion structural proteins of cytoplasmic polyhedrosis virus （CPV） are composed of capsid shell protein VP1, turret protein VP3 and large protrusion protein VP5, which are encoded by genomic segment 1, segment 4 and segment 7 of Cypovirus, respectively. This paper aimed to study the localization of the polyclonal antibodies of structure proteins on CPV virions surface, so as to understand the immunolocalization of CPV structural proteins. Dendrolimus puntatus cytoplasmic polyhedrosis virus （dsRNA） genome was extracted and the full-length and partial fragments of genomic segment 1, segment 4 and segment 7 of DpCPV were reversely transcribed, cloned and expressed in E. coli. The polyclonal antibodies against the expressed proteins were raised in rabbits. Western blot analysis indicated that DpCPV structural proteins VP1, VP3 and VP5 were encoded by S1, S4 and S7 respectively. The immune colloidal gold assays ensured that the structural proteins VP1, VP3 and VP5 localized on the surface of the viral particle. By immunology methods, we firstly determined the localization of CPV structural proteins on virions surface. The results will help to understand the functions of structural proteins and infection mechanism of DpCPV virions.