传染性支气管炎病毒青岛腺胃分离株(SD/97/02)S2蛋白基因的序列测定和分析

根据GenBank上发表的IBV S2基因序列,自行设计了两对引物,分别扩增SD／97／02株S2基因的上游1100bp部分和下游的900bp部分,两段序列拼接后包含了S2全基因的序列。将此两段分别克隆入pGEM T-easy载体,测序后将序列用分析软件DNASTAR和网上的BLAST软件进行分析,结果表明,S2基因比S1基因相对来说更保守,位于氨基酸第560－600位很有是与囊膜锚着的部位,而S2与S1的交界处以HRRRR为特征,这不同地常规的RRF／SRR。     

饲喂重组鸡白细胞介素18蛋白增加肉仔鸡体重的初步研究

经口服途径给肉仔鸡饲喂重组鸡白细胞介素 1 8(ChIL -1 8)蛋白,观察其在正常饲养条件下和人工感染鸡传染性支气管炎病毒 (IBV)时对肉仔鸡的影响。将 84只肉仔鸡随机分为 2组:正常称重组和IBV接种组。 2组肉仔鸡再随机各分为 3个亚组,分别每隔 5d各口服 1次PBS、细菌蛋白和重组ChIL -1 8蛋白。正常称重组每亚组 8只,每次口服前称重。IBV接种试验组每亚组 2 0只,在 30日龄时滴鼻接种IBVM41株并每隔 5d称重 1次。各亚组试验鸡分别在正压隔离器中饲养。结果表明,饲喂重组ChIL- 1 8蛋白的试验亚组肉仔鸡体重总增重量明显高于饲喂PBS和细菌蛋白的对照亚组;IBV接种试验组肉仔鸡在人工感染IBVM41株后,饲喂重组ChIL 1 8亚组的发病率和死亡率明显低于饲喂PBS和细菌蛋白亚组,而且后 2个亚组肉仔鸡临床症状也较饲喂重组ChIL- 1 8试验亚组明显。结果初步显示,饲喂重组ChIL -1 8融合蛋白能够增加肉仔鸡体重并可增强肉仔鸡对IBV感染的抵抗能力。     

鸡传染性支气管炎病毒E蛋白基因的表达

鸡传染性支气管炎(Infectious Bronchitis,IB)是由传染性支气管炎病毒(Infectious Bronchitis Virus,IBV)引起的鸡的一种急性、高度接触性传染病.该病是危害全球养禽业的重要传染病之一[1,2].其感染的主要特征是气管啰音、咳嗽和打喷嚏.此外,该病还可以引起蛋鸡产蛋量下降和蛋品质下降.雏鸡可由于呼吸道或肾脏的感染而死亡.虽然疫苗的使用对IB的流行起到了一定的预防和控制作用,但由于IBV血清型众多,不同血清型毒株之间具有较小的交叉保护性甚至无交叉保护性.因此,IB目前仍在免疫鸡群和非免疫鸡群发生和传播,给养禽业造成了严重的经济损失.     

IBVS1基因表达载体PG-S1的构建

以含有鸡传染性支气管炎病毒S1基因的载体P^IBV-Z和含有CMV启动子的栽体质粒P^EGFR-C1为材料,构建了可表达IBV S1基因的表达载体P^G-S1,经酶切、电泳和PCR检测结果证实,构建的载体符合目的要求,可以作为表达载体用于鸡的抗病育种。     

鸡传染性支气管炎病毒H株纤突蛋白S1基因的克隆及其在毕赤酵母中表达

鸡传染性支气管炎病毒(IBV)河南分离株H,经SPF鸡胚增殖,差速离心纯化病毒,SDS-蛋白酶K法抽提病毒RNA。参照IBV Bcaudette株纤突蛋白S1基因序列设计并合成引物,以其进行RT—PCR,成功地扩增出IBV H株S1基因。扩增产物经Bst YⅠ Hae Ⅲ和Pst Ⅰ酶切分析,结果表明,IBV H株S1基因的RFLP图谱与M41株S1基因的完全一致,初步断定IBV H株为Mass血清型。将IBVH株S1基因克隆于pGEM—T载体中进行序列分析,结果表明该基因全长为1611bp(从ATG到S前体蛋白裂解位点),与标准株M41和Beaudette的S1基因序列相比较,同源率分别达到97．39％和97、27％。将IBV H株S1基因亚克隆到pPICZ—A表达载体,转化毕赤酵母中,SDS—PAGE实验证实了IBV H株S1基因在毕赤酵母中得以表达,进一步用鸡抗IBV血清做Westernblot检测．证实了表达产物的抗原特异性。     

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166–247 aa, S1 gene; 501–515 aa, S1 gene; 8–30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD₄₅₀ value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.     

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.     

小鼠肝炎病毒RT-PCR检测方法的建立和比较研究

用小鼠肝炎病毒的MHV1、MHV3、A59、JHM四株分别感染DBT细胞 ,收获病毒 ,提取病毒RNA。依据小鼠肝炎病毒的病毒基因、M结构蛋白、mRNA的基因保守区分别设计出多对引物 ,按各对引物的条件建立了RT -PCR方法 ,比较了不同引物敏感性和特异性 ,并比较选择与MHV同为冠状病毒的传染性支气管炎 (IBV)标准株和野毒株没有交叉的最佳特异引物。敏感性实验检测到 1 0pg的小鼠肝炎病毒RNA。     

用RT-PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型

用ＲＴ－ＰＣＲ方法获取了禽传染性支气管炎病毒（ＩＢＶ）标准毒株Ｍ４１、Ｈ５２、Ａ５９６８和国内分离株Ｄ４１、Ｆ、Ｇ的Ｓ１基因,对它们做ＲＦＬＰ分析,发现Ｄ４１、Ｆ株属于马萨诸塞血清型、Ｇ株为变异株。对用ＲＴ－ＰＣＲ和ＲＦＬＰ来区分ＩＢＶ的分型方法的应用理论和前景进行了论述。