【作者】 罗丹丹；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 对本实验室鉴定的鸭瘟病毒UL47基因(GenBank NO.EU195109)进行生物信息学分析。针对UL47全基因和UL47基因主要抗原域分别设计并合成特异性引物,进行PCR扩增及序列测定。原核表达并制备了UL47基因主要抗原域(UL47基因ORF的1417-2353bp)蛋白的多克隆抗体,对UL47进行了转录时相研究,获得如下结果：DPV UL47基因的ORF为2367bp,由788个氨基酸组成,分子量约为87.95kDa,等电点为6.23,亲水、疏水氨基酸分别为227个和242个,各占28.8%和30.7%,酸性、碱性氨基酸分别为106个和121个,各占13.5%和15.3%。疏水性预测表明UL47蛋白疏水性最大值为2.6,最小值约为-3.8,疏水区主要位于369-370,393-406,490-493,510-516,526-528,622-630和641-648位氨基酸,亲水区在多肽链占据的位置明显大于疏水区。该多肽有73个潜在的磷酸化位点和3个N-糖基化位点,但无信号肽和跨膜区。亚细胞定位分析表明,UL47蛋白有69.6%分布于细胞核,13%分布于细胞膜,还有少量分布于线粒体、细胞浆和囊泡分泌系统中。二级结构预测结果显示该蛋白以a-螺旋为主(40.36%),β-转角和无规则卷曲的含量较少,都为11.93%。利用EMBOSS的CHIPS和CUSP程序分析DPV UL47基因密码子,结果表明其CAI、ENC值分别为0.71和52.40。由六种密码子编码的Arg,Ser和Leu,分别偏爱AGA,TCT和CTC,其Frequency(1/1000)高达24.081-31.686‰,而TCA、CTA的使用频率为零。Val偏爱GTC,Ile偏爱ATC,Phe偏爱TTC,His偏爱CAT,Gln偏爱CAA,三个终止密码子出现的频率较高。DPV UL47基因整体密码子的使用情况与其他(大肠杆菌、酵母和人)的差值较小,不具有明显偏差。另外,DPV UL47 G+C含量为44.99%,而第三位密码子的G+C含量(即GC3s)就达51.58%,表明UL47在第三位密码子上偏爱使用G或C。稀有密码子分析表明DPV UL47基因有72个稀有密码子,占总密码子的3.04%,其中21个AGA,13个AGG和6个CGA为精氨酸稀有密码子,12个CTA为亮氨酸稀有密码子,6个ATA为异亮氨酸稀有密码子,4个CCC为脯氨酸稀有密码子。系统发生树分析表明DPV UL47更接近a疱疹病毒UL47基因,并且与GaHV-2, GaHV-3, MeHV-1和MDV-2等禽类疱疹病毒的进化关系最近,以上分析结果为开展UL47基因功能研究具有一定的参考作用。通过对UL47全基因和UL47基因主要抗原域分别设计引物,进行PCR扩增及构建T克隆和亚克隆重组质粒,通过BamHⅠ单酶切、BamHⅠ/XholⅠ双酶切、质粒PCR及测序鉴定,成功将全基因及主要抗原域片段分别定向插入载体中,构建了T克隆及亚克隆重组质粒。IPTG诱导BL21进行表达。结果表明,UL47全基因未能表达,而UL47主要抗原域成功表达,蛋白大小为55kDa,符合理论值。表达形式分析表明UL47融合蛋白为包涵体,大量存在于超声破碎后的沉淀中。经过一系列表达条件优化表明37℃下,加入0.2 mmol/L的IPTG诱导6h,UL47主要抗原域蛋白表达量最大。利用纯化后的UL47主要抗原域蛋白制备兔抗UL47多克隆抗体,琼扩效价为1：16。免疫印迹结果显示UL47主要抗原域蛋白能与兔抗DPV抗体和兔抗UL47抗体相互作用,表明该蛋白具有免疫原性。此外,根据UL47基因一段核苷酸片段设计5’端以地高辛标记的寡核苷酸探针,然后应用该探针对GPV、DHV、PRV、MDV、MDPV、ILTV、DPV以及UL47基因T克隆质粒进行检测,同时设置未接毒的DEF核酸提取物为阴性对照,与实验组平行操作。试验结果为UL47基因T克隆重组质粒和DPV DNA显示紫褐色,其余均不显色,表明UL47基因确为DPV CHv UL47基因。根据内参基因β-actin和鸭瘟病毒UL47基因的保守序列分别设计了荧光定量引物,收集感染鸭瘟病毒后1h、2h、4h、6h、8h、10h、12h、14h、16 h、20 h、24h、30 h、36h、48h、54h、60 h、72h等不同时间段的鸭胚成纤维细胞,采用Trizol法提取细胞总RNA,然后进行逆转录,以cDNA为模板进行荧光定量PCR扩增。试验结果显示感染鸭瘟病毒后的8h以内,UL47基因一直处于低转录水平,24h后转录产物量出现较迅速增加,36h达到峰值,随后产物量有所下降,但仍持续到感染后72h。更多还原

【Abstract】 This article concentrates on the UL47 gene (Genebank No. EU195109) of Duck Plague Virus. The bioinformatics analysis of DPV UL47 gene was studied first. Then, we designed two pairs of primers for the full-length UL47 gene and UL47 gene major antigen determinant sequence, amplified them by PCR and did the prokaryotic expression, respectively. We expressed protein of UL47 gene major antigen determinant sequence successfully and utilized the protein to prepare polyclonal antibodies.The biological characteristics of DPV UL47 gene were also carried out later. The results are as follows:The UL47 gene of DPV was composed of 2367 base pair, encoding for a polypeptide of 788 amimo acid, with a molecular mass 87.95kDa. There were 106 acidic amino acids (13.5%),121 basic amino acids (15.3%),242 hydrophobic amino acids (30.7%) and 227 hydrophilic amino acids (28.8%) in the polypeptide. The predicted isoelectric point was 6.23. The polypeptide had 73 potential phosphorylation sites and three potential N-linked glycosylation sites. In DPV UL47 protein, the main hydrophobic region were between aa 369-370,393-406,490-493,510-516,526-528,622-630 and 641-648. In addition, UL47 protein had no transmembrane domain and signal peptide. Subcellelar localization preduction showed that the UL47 protein mainly concentrated in nuclear (69.6%), the rest were existed in plasma membrane (13%), cytoplasm, mitochondrial and vesicles of secretory system. The secondary structure prediction of DPV UL47 protein indicates the DPV UL47 protein consists of 40.36% alpha helix,11.93% Beta turn and 11.93% random coil.The EMBOSS CHIPS and CUSP programs were used to deduce the CAI, ENC value and GC3S content of DPV UL47 gene, and the results were 0.71,52.40 and 51.58%, respectively. A high level of codon usage bias existed for encoding the selection of Leu, Arg, Gly, Val, Ser, His, Ala, Pro. And an extremely low level of codon usage bias existed for encoding the selection of Leu, Ser, Ala and Pro. Rare codons analysis showed that there were 72 rare codons in DPV UL47 gene. Twenty-one AGA codons, thirteen AGG codons and six CGA codons, which are the rare Arg codons. Twelve CTA codons are the rare Leu codons. Sixteen ATA codons are the rare Ile codons. Four CCC codons are the rare Pro codons. Multiple sequence alignment of the amino acid sequences and phylogenetic tree analysis demonstrated that the DPV UL47 and some fowl herpesviruses such as GaHV-2, GaHV-3, MDV-2 and MeHV-1 were clustered within a monophyletic clade and as a result it has a close evolutionary relationship with alphaherpesviruses.We used PCR primers to amplify the UL47 gene and its major antigen determinant sequence, respectively.Then the products was directionally inserted into pMD18-T and pET-32a(+) vector to construct prokaryotic expression system. The recombinant plasmids of T-cloned and sub-cloned were identified by PCR, restriction analysis and sequencing. Then the sub-cloned plasmid pET-32a (+)/UL47 was transformed into BL21 and expressed by IPTG introduction. SDS-PAGE showed that a distinct band of approximately 55kDa molecular weight, corresponding to the expected size, but we didn’t observe a distinct band of approximately 108kDa molecular weight appearing in the corresponding position. These results may confirm that the full-length UL47 gene couldn’t be expressed.Purified protein was used to immunize rabbit for the UL47 anti-serum preparation, and the antibody titer was up to 1:16 by agar diffusion reaction. Western blot analysis using the resultant sera showed that the recombinant protein was recognized by the polyclonal antibody. Thus, the polyclonal antibody prepared here may serve a good tool for study of the functional involvement of UL47 in the DPV life cycle. For dot blot analysis, one digoxigenin-labeled DNA probe of UL47 gene was designed. To test species specificity, the recombinant plasmid pMD18-T/UL47, DPV, GPV, PRV, DHV, ILTV, MDPV and MDV were extracted and analyzed by the dot blot assay. Non-infected DEF served as the blank control. The results showed that the DPV DNA and pMD18-T/UL47 gave a very distinct color signal, and others showed no signals. It implied that the UL47 gene existed only in DPV.The phase analysis of DPV UL47 gene transcription was detected by FQ-PCR method. The results showed that the transcripts of UL47 gene slowly rised up at the first 8 hours post-infection (hpi), then increased rapidly with the extension of the infection time and reached a peak at 36 hpi, lowed down slowly but still detected the transcripts of UL47 gene until 72 hours after infection.更多还原