对虾白斑综合征病毒结构蛋白基因vp95表达时序分析及其抗体的制备

Transcriptional Analysis of the vp 95 Gene of White Spot Syndrome Virus and Preparation of Polyclonal Antibody of the Structural Protein

对虾白斑综合征病毒（whitespotsyndromevirus，WSSV）是造成养殖对虾病害的主要病原，其300kb的双链环状DNA基因组共编码532个开放阅读框（openreadingframes，ORF）．蛋白VP95是WSSV的结构蛋白，在病毒囊膜蛋白和核衣壳中均有分布，由ORFwsv442编码，基因全长2400bp．首先对基因vp95的表达时序进行分析，证明该基因属于晚期基因．为了更深入地研究该结构蛋白的功能，选择中间编码200个氨基酸的片段（1051～1650nt）进行克隆表达．此片段被克隆到pET-His载体上，构建好的质粒转化入表达菌株大肠杆菌（Escherichiacoli）BL-21（DE3），经IPTG诱导后，收集细菌，超声破碎后，将含有可溶性表达重组蛋白的上清用Ni2＋ -NTAagaros纯化．纯化的重组蛋白HisVP95-M200免疫小鼠，制备抗血清．为了去除血清中的杂质，用HiTrapNHS-activatedHP（GEHealthcare）亲和层析纯化柱子纯化血清中抗蛋白VP95的抗体．Westernblot分析显示，未纯化的血清和纯化的抗体对病毒粒子中蛋白VP95都有很高的效价，并且具有严格的专一性．为进一步研究VP95蛋白在病毒粒子的包装中的作用，以及其在病毒的入侵过程中的功能奠定了良好的基础．

White spot syndrome virus(WSSV))is the largest known animal virus,which has a 300kb circular double-strand DNA genome encoding 532open reading frames(ORF).VP95is a structural protein of WSSV which encode by ORF WSV442of 2 400 nucleotides.In present study,a time course analysis showed that this transcript was actively transcribed at the late stage.To investigate this structural protein,apart of vp95 gene(1 051-1 650nt)was cloned from WSSV genome and then was cloned into pET-His prokaryotic expression vector.The recombinant vector was transferred into Escherichia coli strain BL-21(DE3)and the transferred E.coli were induced by IPTG in order to acquire recombinant fusion protein which was subsequently purified by Ni 2+-NTA agaros. The purified protein was used as antigen to immune the rats.To remove the impurities in the serum,we have chosen the prepackaged column HiTrap NHS-activated HP(GE Healthcare)for preparative affinity chromatography.The immunological specificity of both the unpurified serum and the purified antibody were validated by western blotting.This study has provided an important foundation for further study of the role of VP95in the package of virion and its function in the viral invasion.