摘要
论文对本实验室鉴定的鸭瘟病毒(DPV)UL27基因(GenBank登录号EF608147)开展了以下系列研究:DPV UL27基因生物信息学分析及多肽抗原表位预测,主要抗原域的克隆、原核表达和抗体制备纯化及以此为基础的亚细胞定位,运用免疫印迹和荧光定量方法分析表达实相和转录实相。应用表达蛋白制备亚单位疫苗进行攻毒保护试验,结果如下:
1.采用软件DNAStar Protean程序,综合运用二级结构、亲水性、可塑性和抗原性指数等参数,对DPV CHv毒株UL27基因(GenBank登录号EF608147)的氨基酸序列进行B细胞表位预测,分析结果显示在位于UL27蛋白羧基端第aa151-550区域内含有主要抗原决定簇。
2.以DPV CHv毒株基因组为模板,用primer5.0软件设计一对引物,整体扩增涵盖具有优势表位的基因片段(451-1650bp),进行原核表达。用原核表达产物作为免疫原制备兔抗DPV多克隆血清,以此抗血清进行Western blotting检测显示抗血清可与该融合蛋白发生结合反应。利用重组表达蛋白所带的6×His标签用镍柱亲和层析两种方法对其进行纯化,获得较高纯度的重组蛋白(UL27M)。过柱纯化蛋白分别对家兔进行免疫,制备兔高免血清,ELISA效价为1:819200,具有中和活性。
3.通过免疫荧光对病毒感染DEF的亚细胞定位发现特异性荧光可最早在感染后8h的核膜上检测到,12h核膜周围荧光数量增多,24h荧光数量达到一个相对高的水平并向胞浆转移,36h荧光基本分布胞膜边缘,48h荧光分布于细胞各个区域。
4.鸭瘟强毒体外感染鸭的宿主细胞UL27基因转录及表达时相分析。荧光定量PCR分析结果显示DPV UL27基因转录产物最早出现于感染后6h,于感染后14h达到高峰,之后一直维持到60h。这种转录谱具有疱疹病毒晚期基因的典型特征。以制备的兔抗UL27M蛋白IgG为一抗,对感染鸭瘟强毒的DEF进行Western blot分析结果显示在感染60～72h之间的细胞蛋白提取物中可观察到与DPV UL27蛋白预测大小约110kD相一致的条带,在感染后60h达到最大值,一直持续到感染后72h。基因转录及表达时相分析推测DPV UL27基因可能是晚期基因。
Base on duck plague virus(DPV) UL27 gene(GenBank accession number EF608147) to carry out the following series of studies:Bioinformatics Analysis of DPVUL27 and predicted epitope peptides.The main antigen domain cloning, prokaryotic expression,preparation of polyclonal antibody and Subcellular localization.Using Western blot and fluorescence quantitative PCR analysis time course of UL27gene expression and transcription.Prepare subunit vaccine using UL27-M proteinum and carry out experiment of infect protection.The results are as follows:
1.Using DNA Star Protean software,the UL27 gene amino acid of the duck plauge virus(DPV) CHv strain(GenBank accession number EF608147) was analyzed on secondary structure and hydrophilicity and antigenic index,with the B cell epitopes to be predicted.The results showed that the epitopes of UL27 were situated in aa151-550,
2.The genomic DNA of DEV CHv was extracted as PCR template,One pair of specific primers was designed by using primer5.0.A fragment coding for UL27 major antigen epitopes(451-1650bp) was amplified by PCR technique and was expressed by prokaryotic expression.Western-blot analysis indicated that multiclone anti-serum of DPV had specific reaction with the recombinant protein.The expression product was purified successfully by passing the Ni~+ affinity chromatograph Collumn using the recombinant protein with a tag of 6×His.The rabbit was immunized with the purified protein,and ELISA showed that the valence was up to 1:819200,has neutralize activity.
3.Immunolocalization of DPV gB gene products in virus infected DEF Immunolocalization detection was observed using immunofluorescence technique, results shown that specific fluorescence appeared on nuclear membrane as early as 8hours post infection and a great deal of specific fluorescence concentrated in the cell nucleus by 12 hours,24h fluorescence maximum and transfer to the cytoplasm,36h fluorescent almost around cell membrane,48h fluorescent distribute all regions.
4.Time course of gene products in DPV infected host cells and transcriptional analysis.Fluorescence quantitative PCR shows the DPV UL27 gene transcripts appeared as early as 6h post infection,then Fluorescence signal intensity increased steadily and reached a peak at 14 h,and remained detectable up to60 h,which owes the typical characterization of herpervirus late genes.A time course of DPV infected DEF were analyzed by western-blot using the polyclonal antibody IgG against DPV UL27M.A specific immunoreactive band migrating was observed at the expected position for protein DPV gB(about 110kDa) with maximal amounts between 60 and 72h.
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