鸭肠炎病毒UL18基因的转录、表达特征及原核表达蛋白的初步应用

Transcription and expression characteristics of duck enteritis virus UL18 gene and preliminary application of prokaryotic expression protein

为了探讨鸭肠炎病毒（duck enteritis virus，DEV）UL18基因的特性和功能，本研究运用生物信息学软件对DEVUI，18基因及其编码蛋白进行了分析。构建原核表达质粒pET32a-UL18，并将其转化大肠杆菌BL21（DE3），经IPTG诱导后获得了大小约为55000的pET32a／DEV-UL18重组蛋白。将该重组蛋白纯化后免疫家免，获得了兔抗pET32a／DEV-UL18重组蛋白高免血清。采用荧光定量RT-PCR和Westernblot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检测，采用间接免疫荧光对DEV感染鸭的肠道和食道进行检测。生物信息学分析结果表明，DEVUL18基因大小969bp，编码1条由322个氨基酸残基组成的多肽，含15个潜在的磷酸化位点和2个N-糖基化位点，合15个潜在的B细胞表位；系统进化树分析表明，DEVUL18属于＆一疱疹病毒的1个成员，与MeHV-1亲缘关系最近；密码子偏爱性分析结果表明，若对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，则需对宿主表达菌进行选择和条件优化。荧光定量RT-PCR和westernblot结果表明，DEV感染DEF后2h，UL18基因开始转录，感染后12h开始表达，感染24h后转录和表达产物急剧上升，到36和48h后转录和表达产物分别达最大值，之后逐渐下降。间接免疫荧光结果表明，被DEV感染鸭肠道、食道等组织能检测到明显的阳性信号，表明制备的DEVUL18原核表达蛋白及其兔抗多克隆抗体可用于DEV的诊断试剂材料。

To investigate the characteristics and function of duck enteritis virus （DEV） UL18 gene, the DEV UL18 gene and its encoded protein were analyzed using bioinformatics analysis software. The prokaryotic expression plasmid pET32aUL18 was constructed and transformed into E. coli BL21 （DE3）. After induction by IPTG,recombinant pET32a/DEVUL18 protein with a size of ap proximately 55 000 was obtained. After the recombinant protein was purified,the rabbit was im munized to obtain rabbit antipET32a/DEVUL18 hyperimmune serum. The transcription andexpression of DEV UL18 gene in DEVinfected duck embryo fibroblasts （DEF） was detected by fluorescent quantitative RTPCR and Western blot. The intestine and esophagus of DEVinfected ducks were detected by indirect immunofluorescence. Bioinformatics analysis showed that the DEV UL18 gene is 969 bp in size and encodes a polypeptide consisting of 322 amino acid residues,con taining 15 potential phosphorylation sites,2 N glycosylation sites,and 15 potential B cell epitopes.Phylogenetic tree analysis showed that DEV UL18 belongs to a member of aherpesvirus and has a close relationship with MeHV1. The results of codon bias analysis showed that if the DEV UL18 gene is expressed exogenously,the eukaryotic expression system may be easier. If the prokaryotic expression system is selected,the host expression bacteria must be selected and the conditions of expression need to be optimized. The results of fluorescent quantitative RTPCR and Western blot showed that the UL18 gene was transcribed at 2 h after DEV infection with DEF,and it began toexpress at 12 h after infection. The transcription and expression products increased rapidly after in fection for 24 h,and the transcription and expression products reached the maximum after 36 h and 48 h respectively, then decreased gradually. The results of indirect immunofluorescence showed that the DEVinfected duck intestinal and esophageal tissues could detect significant positive sig nals,indicating that the prepared DEV UL18 prokaryotic expression protein and its rabbit anti polyclonal antibody can be applied to DEV diagnostic reagent materials.