【作者】 陈希文；

【导师】 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2013， 博士

【摘要】 1.鸭肠炎病毒UL18基因的序列特征与密码子使用偏爱性运用生物信息学分析软件对注册的鸭肠炎病毒(Duck enteritis virus,DEV) UL18基因(GenBank登录号为EU195093)及其编码蛋白进行了分析。结果表明,DEVUL18基因大小969bp,编码蛋白为一条含322个氨基酸残基组成的多肽,相对分子量为35.250KD,等电点理论值为8.37,无信号肽切割位点,含有15个潜在的磷酸化位点和2个N-糖基化位点,含15个潜在的B细胞表位；系统进化分析结果表明,DEVUL18属于α-疱疹病毒的一个成员,与MeHV-1的亲缘关系最近；密码子偏爱性分析结果表明,若要对DEVUL18基因进行外源表达,真核表达系统可能更容易。若选择原核表达系统,则需要对宿主表达菌进行选择和条件优化,才能有利于该基因在体外表达重组蛋白。2.鸭肠炎病毒UL18基因的原核表达、抗体制备及应用根据注册的DEV UL18基因序列,用Primer Premier6.0软件设计合成一对特异性扩增DEVUL18基因的引物。用该引物从DEV基因组中扩增UL18基因,并将其正向插入pET32a(+)原核表达质粒,成功构建了原核表达质粒pET32a-UL18。将pET32a-UL18原核表达质粒转化BL21(DE3)表达宿主菌,以IPTG诱导,获得了大小约为55KD且主要以包涵体形式存在的pET32a/DEV-UL18重组蛋白。将该重组蛋白纯化后免疫家兔,得到了兔抗pET32a/DEV-UL18重组蛋白的高免血清。以纯化的pET32a/DEV-UL18重组蛋白作为包被抗原,建立了基于pET32a/DEV-UL18重组蛋白的间接ELISA方法；以纯化的兔抗pET32a/DEV-UL18重组蛋白IgG作为一抗,建立了基于兔抗UL18重组蛋白IgG的免疫组化和免疫荧光方法,并对DEV感染鸭组织和鸭胚成纤维细胞后UL18蛋白的分布进行了检测。3.鸭肠炎病毒UL18基因在感染宿主细胞中的转录与表达特征采用荧光定量RT-PCR和Western blotting对DEV感染鸭胚成纤维细胞(DEF)后UL18基因的转录和表达情况进行了检测。结果表明,DEV感染DEF后2h,UL18基因已经开始转录,感染后12h检测到开始表达,感染24h后转录和表达产物急剧上升,到36h和48h后转录和表达产物分别达最大值,之后逐渐下降。DEV UL18基因在DEF细胞中的表达产物分子量约为35KD。根据该基因的转录和表达特征,初步推测DEV UL18基因为病毒晚期基因。更多还原

【Abstract】 1. Analysis of duck enteritis virus(DEV) UL18gene for its sequence features and codon bias. The DEV UL18gene sequence (GeneBnak accession EU195093) and its protein sequence were analyzed with bioinformatics software. The results showed that DEV UL18gene was969bp,its encoded protein was a polypeptide which comprised of322amino acids residue, the relative molecular weight was35.250KD, the theoretical value of its isoelectric point was8.37,which had no N-terminal signal peptide, but contained15potential phosphorylation sites,2glycosylation sites,and15potential B-cell epitopes. The phylogenetic tree analysis showed that DEV UL18belonged to one of the Alphaherpesvirinae, which was evolutionarily closest to the MeHV-1.It is proper to choose eukaryotic expression system to express this gene based on the codon analysis.So it might be necessary to screen different host bacteria and optimize the condition if using a prokaryotic expression system to expressing this protein.2. Prokaryotic expression and antibody preparation of DEV UL18gene and its application. The primers were designed based on the DEV UL18gene sequence using software Primer Premier6.0. The UL18gene fragment was amplified from DEV genomic DNA by PCR and was cloned into pMD18-T vector. The recombinant construct, named pMD18-T-UL18, was verified by PCR and double digestion (Hind Ⅲ and Xho Ⅰ) and DNA sequencing. Then the UL18gene fragment was subcloned into the prokaryotic expression plasmid, pET32a(+).The recombinant plasmid pET32-UL18was transformed into the host bacteria BL2](DE3), the optimal concentration was0.6mmol/L IPTG as inductor, the optimal time was4h at37℃, and the recombinant expressed protein is about55KD, which was mostly existed in inclusion body. The recombinant protein was purified, rabbits were immunized four times after immunization, then collected the hyperimmune serum of the rabbit anti pET32a/DEV-UL18. Based on the purified recombinant protein pET32a/DEV-UL18, DEV-UL18-ELISA was developed for detecting the DEV serum antibodies in an indirect ELISA. Indirect immunoperoxidase assay (IPA) and indirect immunofluorescence assay (IFA) with the rabbit IgG raised against UL18recombinant protein was used to study the distribution in the tissues of infected ducks.3. Transcription and translation characteristics of DEV UL18gene in infected host cells. UL18mRNA and protein were detected through qRT-PCR and western blotting respectively. The result showed that the transcription of DEV UL18were started at2h post infection (PI),and the detection of its expression products were at12h PI,the transcripts and expression products were surging at24h PI,afterwards came to a peak at36h and48h PI,then both of them were reduced.The expression products was nearly35KD. It can be inferred that this gene was in the last period of the virus by analyzed synthetically the characteristics of DEV UL18’s transcripts and expression.更多还原