黄病毒E蛋白结构域Ⅲ能够介导病毒与宿主受体结合以及诱导产生中和抗体,本研究利用原核表达系统表达了鸭坦布苏病毒E蛋白结构域Ⅲ,并研究了其免疫原性。按照大肠杆菌偏好性密码子对鸭坦布苏病毒E蛋白结构域Ⅲ核苷酸序列进行优化并合成后,克隆至pCold-TF载体构建重组质粒pCold-TF-optiEDIII。Western blot证实重组蛋白能与鸭坦布苏病毒特异性血清发生反应。用纯化的重组蛋白免疫BLAB/c小鼠3次,间接ELISA和间接免疫荧光实验证实E蛋白结构Ⅲ诱导了鸭坦布苏病毒特异性的体液免疫反应。中和实验证实鸭坦布苏病毒E蛋白结构域Ⅲ可诱导产生中和抗体。本研究为进一步研制鸭坦布苏病毒诊断抗原和亚单位疫苗奠定了基础。

The domain III of envelope glycoprotein (E) of the flaviviruses has been shown to bind cellular receptors and elicit neutralizing antibodies. In the present study, recombinant domain III (DIII) of E protein of Duck Tembusu virus (DTMUV) was expressed in E.coli and its immunogenicity was evaluated using BALB/c mouse model. The coding sequence of DIII was optimized (optiDIII) according to the bias codon usages of E.coli and then cloned into the multiple cloning site of pCold-TF vector to construct the recombinant plasmid pCold-TF-optiDIII. Western blot analysis showed the recombinant protein could react specifically with DTMUV antiserum. The recombinant DIII was purified and used to immunize BALB/c mice three times. The humoral immune responses specific for DTMUV were demonstrated by ELISA and indirect immunofluorescene assay(IFA). Importantly, neutralizing antibodies against DTMUV were detected in mouse serum samples by microneutralizing test. These findings have indicated that the reco

鸭坦布苏病毒是近年来在我国发现的可引起种（蛋）鸭高热、采食量和产蛋量骤降的新病毒。本研究以麻鸭为动物模型，测定麻鸭感染鸭坦布苏病毒后，其免疫器官指数、重组H5亚型禽流感病毒灭活疫苗和鸡新城疫病毒弱毒活疫苗诱导产生的抗体水平。结果表明，鸭坦布苏病毒感染会侵害麻鸭的免疫器官，第1～3d脾脏明显肿大，第7～10d试验组胸腺指数极显著低于对照组，第1～14d法氏囊出现萎缩；与仅免疫疫苗的对照组鸭相比，感染鸭坦布苏病毒后再注射疫苗的试验组鸭产生抗体的时间延迟、抗体的峰值降低，抗体下降加快，且在整个试验期内产生的抗体水平均低于对照组鸭的抗体水平，表明鸭坦布苏病毒感染可抑制鸭对灭活疫苗和弱毒疫苗的体液免疫应答能力。

Duck Tembusu virus is a newly discovered virus in recent years ,which caused duck high fever ,egg-laying and feed intake decreased .In this study ,we used Sheldrake as animal model ,and infected with duck Tembusu virus .Then we determinated the immune organ indices and the antibody levels in ducks induced by recombinant H 5 avian influenza inactivated vaccine and Newcastle disease virus attenuated live vaccine , respectively .The results showed that the duck immune organs were violated after infected with duck Tembusu virus ,resulting in spleen enlargement (1 dpi to 3 dpi) ,bursa atrophy (1 dpi to 14 dpi) ,and the test group thymus index was significantly lower than the control group from 7 dpi to 10 dpi .Compared with immunized-group ,the antibodies level of the infected-immunized-group were delayed and low ,and reduction in peaks and declined accelerate throughout the test period .The above results indicated that duck Tembusu virus can affect the humor immune response to inactivated

为明确禽坦布苏病毒包膜蛋白E的基因特征，运用RT-PCR从已经分离鉴定的禽坦布苏病毒中扩增出其包膜蛋白E全基因，并进行克隆测序和分析。结果表明，禽坦布苏病毒包膜蛋白E全长为1503bp，编码501个氨基酸。所编码的包膜蛋白E大小为54.217ku，理论等电点为6.89，不稳定系数为25.48，属于稳定蛋白质类。利用TMHMM2.0进行跨膜区分析发现，该蛋白的拓扑结构为o442-464i471-489o。本试验株包膜蛋白E和北京鸭源坦布苏病毒（GenBank号：JF459991）同源性最高，达99.8％，和雌麻鸭源坦布苏病毒（GenBank号：JQ928189）核苷酸同源性最低，为98.2％，不同来源（鸡源、鸭源、鹅源、鸽源和蚊子源）坦布苏病毒包膜蛋白E同源性均较高，没有表现出宿主特异性。

The envelop protein fragments of avian Tembusu virus were amplified by RT-PCR from the identified avian Tembusu virus isolated in Fujian ,and cloned to the T-vector for sequenceing and bioinformatics analysis .The results revealed that the avian Tembusu virus envelop gene contained 1 053 nucleotides ,coding an open reading frame (ORF) with 501 amino acids .The molecular weights ,theoretical isoelectric point and the instability index of avian Tembusu virus envelop protein were 54.217 ku ,6.89 and 25.48 ,respectively .The topology predicted by N-best using the TMHMM2.0 is o442-464i471-489o .The sequenced envelop protein shared the highest homogeneity with Peking duck-origin avian Tembusu virus strain (GenBank accession number is JF459991) ,which was 99.8% , while shared with common sheldduck-origin avian Tembusu virus strain (GenBank accession number is JQ928189) 98.2% homogeneity .The results demonstrated that avian Tembusu virus had no host specificity.

种番鸭是否感染禽坦布苏病毒颇受国内学者关注。本研究于国内外首次自表现产蛋下降的种番鸭卵巢中分离获得1株病毒(命名为WYZLJ-1359株),经RT-PCR方法鉴定为禽坦布苏病毒。经比较分析该病毒与我国其他禽源分离株全基因组序列表明,种番鸭源分离株WYZLJ-1359主要分子特征未出现变异,与2010年以来我国分离的其他禽源分离毒株的核苷酸序列同源性均在98.4%以上,且亲缘关系非常近,遗传距离均在1.8%以下,远低于与2000年以前分离的坦布苏病毒分离株之间的遗传距离。以上结果表明,我国禽坦布苏病毒感染的宿主在不断增加,但病毒在不同品种家禽的传播过程中遗传较为稳定,基因组未出现基因的缺失或插入等变异现象。

Nowadays ,it is still unknow whether the avian tembusu virus could infect breed Muscovy duck .For the first time ,we obtained a viral isolate (named WYZLJ-1359) from ovary of breed Muscovy duck with decreasing egg-laying rate ,and confirmed the infectious agent as avian tembusu virus (ATV) using RT-PCR .Results of sequence comparison with viral genome showed that the main molecular characteristics of genome sequence of WYZLJ-1359 strain were in accordance with those of other viruses isolated from other domestic poultry since 2010 ,and shared no less than 98.4% nucleotide sequence identity with each other .Phylogenetic analysis showed that all ATVs isolated since 2010 shared closed relationship with each other .And the genetic distance (GD) among these viruses were all no more than 1.8% ,which was far less than the GD between WYZLJ-1359 strain and those viruses reported before 2000. These results indicated that although the host spectrum of avian tembusu virus continued to increase ,the

利用RT-PCR方法扩增鸭坦布苏病毒分离株(WR株)全长E蛋白基因,克隆到pEASY-T3载体中,经Bam HI和XhoI酶切后将目的片段连接入pET-32a载体,构建原核表达重组质粒pET-E。表达质粒转化入感受态细胞BL21(DE3)后,经IPTG诱导后表达出鸭坦布苏病毒E蛋白,并以包涵体的形式存在,Westernblotting试验呈阳性,表明E蛋白有很好的反应原性。

The entire E gene from the duck Tembusu virus (strain WR)was amplified by RT-PCR,and cloned into thepEASY-T3 vector.The recombinant plasmids carrying the target fragment were digested with Bam HI and Xho I,andcloned into the pET-32a vector to construct recombinant plasmid to be named pET-E.pET-E was subsequently transformedinto Escherichia coli BL21 (DE3 ).The fusion protein was expressed by IPTG induction,and presented mainly asinclusion bodies.The positive western blotting result suggested a strong reactinogenicity of the protein.