Expression,Purification and Activity Detection of Structural Protein VP1 of Foot-and-Mouth Disease Virus Serotype A

The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.

Establishment of Indirect ELISA Diagnosis Technique based on the VP1 Protein of Foot and Mouth Disease Virus Serotype A

The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.

Asia1型口蹄疫病毒多表位基因的原核表达及ELISA检测方法的建立

根据国内流行的Asia 1型口蹄疫病毒的基因组特性,合成了Asia 1型口蹄疫病毒2个流行毒株VP1基因的5个抗原表位,并将其克隆到pMD18-T载体上,再按设计的酶切位点连接,构建出Asia 1型口蹄疫病毒VP1双拷贝基因片段(VP1 Asia)。然后,将VP1 Asia基因连接到原核表达载体pET-28a(+)上,构建了重组表达质粒pET-28a-VP1 Asia,接着将其转入BL21菌中进行原核表达。以纯化的表达蛋白作为抗原检测了牛Asia 1型口蹄疫病毒阳性血清。结果显示,VP1 Asia基因在大肠杆菌中获得了高效表达,以纯化的VP1Asia蛋白作为抗原建立了检测Asia 1型口蹄疫病毒抗体的ELISA方法。以建立的ELISA方法和标准Asia 1型口蹄疫液相阻断ELISA试剂盒对30份临床样品进行了检测,两种检测方法的阳性率分别为90.0%(27/30)和93.3%(28/30),符合率为89.7%(26/29)。本研究为组装Asia 1型口蹄疫病毒抗体ELISA诊断试剂盒奠定了基础。

Asia 1型FMDV感染BHK-21细胞差异蛋白质点2-DE分析

为研究Asia 1型口蹄疫病毒(FMDV)感染BHK-21细胞后蛋白质组的变化及差异,将FMDV接种于培养的单层BHK-21细胞,同时设未接毒的BHK-21细胞作对照组。待细胞病变后分别提取2组细胞总蛋白借助2-DE技术及PDQuest8.0软件进行细胞蛋白质组二维电泳图谱差异性分析。结果显示感染组有1 457个可见蛋白质点,而对照组可见的蛋白质点为1 427个。感染组蛋白点的表达量与对照组相比,二者具有统计学差异(P<0.05)的蛋白质点472个,其中表达上调251个点,表达下调221个点,新合成蛋白点30个。结果表明FMDV感染BHK-21细胞后引起了细胞蛋白质组表达模式的改变,这种变化是病毒与细胞相互作用的结果。本研究首次利用蛋白质组学技术研究Asia 1型FMDV感染BHK-21细胞后蛋白质组学差异,有助于深入研究Asia 1型FMDV和BHK-21细胞相互作用的分子机制。

云南边境Asia1型口蹄疫监测及病毒vp1基因序列比较

口蹄疫是由口蹄疫病毒引起的主要侵袭偶蹄动物的一种急性热性高度接触性传染病。口蹄疫病毒存在7个不同血清型,病毒VP1蛋白抗原性差异是病毒血清型划分依据,而其编码基因核苷酸序列差异是同型病毒Topotype型或基因型划分依据。采用RT-PCR及O/A/C/Asia1多重RT-PCR技术,对2006年期间自云南边境地区采集境外流动动物组织样品120份,进行口蹄疫病原监测,检出Asia1型口蹄疫病毒阳性样品7份。对阳性样品中病毒vp1基因全序列进行扩增、纯化后,克隆至pMD18-T载体测序,并与已知代表性毒株进行比对及系统发育分析。结果发现:云南边境Aisa1型口蹄疫病毒阳性样品vp1基因核苷酸序列同源性介于83.4%～99.5%,不同分离毒株以15%序列差异作为分型标准,存在1个新拓朴型或基因型Ⅴ,且与已知的4个拓朴型或基因型不同。系统发育分析确定了1个新的遗传谱系Ⅶ。部分VP1蛋白表位关键性氨基酸位点存在变异。

利用Asia1型口蹄疫病毒VP1蛋白的单克隆抗体建立单抗竞争ELISA方法

应用纯化的Asia1型口蹄疫病毒VP1蛋白作为抗原,采用过碘酸钠法标记纯化的单抗,建立了单抗竞争ELISA,检测10份阳性猪口蹄疫血清及200多份阴性血清,试验结果与样品的背景相一致。敏感性试验和重复试验结果表明,该方法具有稳定和敏感等特性,适合Asia1型猪口蹄疫病毒抗体的监测。

共表达Asia1口蹄疫病毒P1-2A-3C基因和猪IL-18基因重组鸡痘病毒构建及表达

为构建Asia1型口蹄疫重组鸡痘病毒疫苗,采集口蹄疫发病牛的水泡液及水泡皮,用RT-PCR法扩增出Asia 1型口蹄疫病毒的前体蛋白基因P1-2A片段和蛋白酶基因3C片段,分别克隆至pMD18-T载体上,通过酶切连接获得质粒pMD18-T-P1-2A-3C。再将P1-2A-3C片段与pUTAL-IL18片段连接起来,构建鸡痘病毒中间转移质粒pUTAL-P1-2A-3C-IL18。通过脂质体转染法,将pUTAL-P1-2A-3C-IL18与鸡痘病毒282E4株共感染染鸡胚成纤维细胞(chicken embryo fibroblasts,CEF),通过BrdU三次加压筛选,筛选出重组鸡病毒株vUTAL-P1-2A-3C-IL18。经RT-PCR和间接免疫荧光法鉴定,证明所筛选的1株重组鸡痘病毒在CEF中能正确表达P1-2A-3C基因。本研究为研制安全、高效的亚洲Ⅰ型FMD重组鸡痘病毒疫苗奠定了基础。

胶体金免疫层析试纸条快速定量检测Asia 1型口蹄疫病毒含量方法的建立

采用胶体金为标记物制备了快速定量检测Asia 1型口蹄疫病毒(FMDV)免疫层析试纸条,拟合检测曲线,用金标分析仪进行定量检测。应用实验室与田间的样品检测证实其有较好的敏感性和特异性。对Asia 1型FM—DV抗原定量检测可以在15 min内完成,灵敏度为0.78μg·mL^(-1),线性范围在0.78~7.84μg·mL^(-1);变异系数在0.2%~5%;回收率在91%~102%,证明定量检测、准确度高。结果表明,本研究建立的定量检测Asia 1型口蹄疫病毒的胶体金免疫层析方法能简便、廉价、快速、灵敏、特异、准确地定量检测样品中的Asia 1型口蹄疫病毒。

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV)can infect domestic and wild cloven-hoofed animals.The non-structural protein 3D plays an important role in FMDV replication and pathogenesis.However,the interaction partners of 3D,and the effects of those interactions on FMDV replication,remain incompletely elucidated.In the present study,using the yeast two-hybrid system,we identified a porcine cell protein,DEAD-box RNA helicase 1(DDX1),which interacted with FMDV 3D.The DDX1-3D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA)in porcine kidney 15(PK-15)cells.DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses.However,the roles of DDX1 during FMDV infection remain unclear.Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner.In addition,DDX1 stimulated IFN-p activation in FMDV-infected cells.Together,our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.

Asia1型口蹄疫病毒VP1蛋白进化踪迹分析

为预测口蹄疫病毒（FMDV）VP1蛋白的重要功能残基,本研究利用进化踪迹及蛋白结构同源建模等方法对91株Asia 1型FMDV的VP1氨基酸序列进行分析,结果显示91个病毒株分属于B、C和D分支,2005年~2006年分离株处于另一个分支,命名为E分支。以10分区分析进化树,共34个组特异残基,其中140S、141S、146L、148A、149L、150A、151R、152R、153V、155N和156R分布于G-H环区域,预测它们与抗原性和受体结合特性有关;B、C、D和E 4个分支的组内特异残基分别为24S、153G、196H;59H、141P、146M、150S、169N;50I、135A和99N、127S、140S、151R,这些残基为各分支的分子标识;组特异残基35V、80L、93S、127A分布于VP1和其他结构蛋白的作用界面,可能对病毒的装配具有重要作用。本研究结果为FMDV的VP1功能研究提供了参考数据。

MDV-1 VP22 conjugated VP2 enhancing immune response against infectious bursal disease virus by DNA vaccination in mice

VP22 of Marek’s disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8+/CD4+ T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P<0.05). Our results demonstrated that VP22 indeed enhances the cell-mediated response in the fused VP2 in a mice model system, possibly due to the fact that the IBDV VP2 could be carried into the surrounding cells at a limited level under pressure from MDV VP22.

Foot-and-Mouth Disease Virus Inhibits RIP2 Protein Expression to Promote Viral Replication

Receptors interaction protein 2(RIP2)is a specific adaptor molecule in the downstream of NOD2.The role of RIP2 during foot-and-mouth disease virus(FMDV)infection remains unknown.Here,our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-βand NF-κB signal pathways during FMDV infection.FMDV infection triggered RIP2 transcription,while it reduced the expression of RIP2 protein.Detailed analysis showed that FMDV 2B,2C,3C^(pro),and L^(pro) proteins were responsible for inducing the reduction of RIP2 protein.3C^(pro) and L^(pro) are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis.The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2.Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2.The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1(PABPC1).The interaction between RIP2 and 2C was observed in the context of viral infection,and the residues 1-61 were required for the interaction.These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.

O型口蹄疫病毒结构蛋白基因VP1的克隆与原核表达(英文)

According to the complete genome of foot-and-mouth disease virus(FMDV)type O,a pair of special primers was designed to amplify VP1 gene.The VP1 gene was amplified by RT-PCR and subsequently inserted into the expression vector pGEX-6p-1 and induced by IPTG.Then SDS-PAGE showed the expressed protein was 51 kD in molecular weight.Then the product was purified by GSTrap FF columns.The product was detected through Western-blot that showed the protein has antigenicity.It provided fundamental data and materials for further investigation on diagnosis method of FMDV.

2013—2019年我国流行性出血病病毒血清1型毒株的分离与遗传特征分析

【目的】掌握流行性出血病病毒(Epizootic haemorrhagic disease virus,EHDV)血清1型毒株(EHDV-1)在我国南方地区的流行情况及其遗传特征,为开展EHDV-1血清型特异性诊断、流行病学调查和致病性研究提供科学依据。【方法】2013—2019年通过在我国云南、广东和广西设立牛羊虫媒病毒监控点和哨兵牛,定期采集哨兵牛血液样本进行虫媒病毒分离;通过微量中和试验确定EHDV分离株的血清型,然后对EHDV分离株的部分基因节段(Seg-2、Seg-3和Seg-6)进行RT-PCR扩增、双向测序及序列分析,并利用BioEdit计算EHDV基因组各节段核苷酸序列及其推导氨基酸序列的相似性。【结果】2013—2019年从云南、广东及广西的虫媒病毒监控点共分离获得33株EHDV,其中7株为EHDV-1型毒株。分离获得的7株EHDV-1型毒株Seg-2、Seg-3和Seg-6核苷酸序列高度同源,其平均相似性分别为(97.65±1.51)%、(94.87±4.72)%和(97.61±1.41)%,对应的推导氨基酸序列相似性平均为(97.69±1.36)%、(99.49±0.32)%和(97.51±2.47)%。基于Seg-2、Seg-3和Seg-6核苷酸序列相似性构建的系统发育进化树均显示,不同EHDV-1型毒株可划分为东方(Eastern)和西方(Western)2种地域型,东方地域型毒株分离自中国、日本及澳大利亚,西方地域型毒株主要来源于美洲和非洲。在基于Seg-2核苷酸序列相似性构建的系统发育进化树中,分离获得的7株EHDV-1型毒株归属于西方地域型,且聚类形成一个相对独立的进化分支;在基于Seg-3核苷酸序列相似性构建的系统发育进化树中,分离获得的7株EHDV-1型毒株分属2种地域亚型(Eastern-1和Eastern-2),分别与日本EHDV-1型和EHDV-6型毒株具有最近的亲缘关系;在基于Seg-6核苷酸序列相似性构建的系统发育进化树中,分离获得的7株EHDV-1型毒株属于东方地域型,与日本EHDV-1型毒株具有较近的亲缘关系。【结论】EHDV-1型毒株已在我国南方地区广泛流行,其Seg-2和Seg-6序列分别具有最近的共有祖先病毒,而Seg-3序列来自不同的祖先病毒。西方地域型毒株Seg-2序列在我国流行EHDV-1型毒株中的出现,说明西方地域型毒株在侵入我国后可能与本土流行的毒株发生重配,即我国流行EHDV-1型毒株为西方地域型与东方地域型的重配型毒株,为防范强致病性西方地域型毒株传入我国而造成畜牧业重大经济损失提供了警示。

Expression and intercellular trafficking of the VP22 protein of CVI988/Rispens vaccine strain of Marek’s disease virus

The viral protein 22 (VP22) in the tegument of Marek’s disease virus serotype 1 (MDV-1) plays an im-portant role in cell-to-cell spread and viral propagation. Antiserum against the carboxyl terminus of VP22 was prepared by immunizing mice with recombinant VP22 expressed in E. coli, and used to in-vestigate its expression in chicken embryo fibroblast (CEF) cells infected with different MDV-1 strains. At an infection dose of PFU=50, intercellular trafficking of the VP22 into the nuclei of the surrounding receipt cells was detected as early as 3 hours post infection. By 6 hours after infection (before viral plague formation), the protein was detected in the whole nuclei of the recipient cells with no difference among MDV-1 strains CVI988/Rispens, GA and RB1B. Intra-nuclear accumulation of the VP22 protein was further increased when the viral plagues started to form. These results indicate that, albeit the ex-istence of the 201TKSERT206 deletion, the VP22 of the CVI988/Rispens vaccine strain has also intercel-lular-trafficking function, which might serve as a potential alternative delivering protein instead of virulent strains VP22.

鸡马立克氏病病毒在鸡淋巴细胞和羽髓中的复制动力学分析

马立克氏病(MD)是由鸡马立克氏病病毒血清1型(MDV1)引起的鸡高度接触性淋巴细胞增生性疾病,MDV1在体内的复制状况与其致病性强弱及传播能力直接相关。本实验选择近几年从国内不同地区MD暴发鸡场分离的6株MDV1强毒株、弱毒疫苗"814"株和国内标准MDV1强毒J-1株,分别人工感染SPF鸡,采用双重实时荧光定量PCR(FQ-PCR)方法,检测感染后1d～28d病毒在淋巴细胞和羽髓中的复制状况。结果显示,接种1d后即可在淋巴细胞中检测到MDV1(102.6copies～105.2copies/106cells);在检测期间,淋巴细胞中病毒载量略有上升的趋势,总体呈现不规律变化,而且变化并不明显。接种7d后羽髓中病毒载量开始显著增加,14d～21d达到峰值,超强毒株峰值处病毒载量可达到107copies/106cells,峰值期病毒载量是感染前期(1d～7d)的100～10000倍。强毒株在体内的病毒载量高于弱毒株,即复制能力高于弱毒株。研究表明,MDV1国内流行的毒株有增殖速度快,病毒载量高的新特点;MDV1致病性的高低与在其在体内的复制能力的高低呈正相关。

由反转录病毒载体转移的马立克病毒糖蛋白D基因在感染细胞中的表达

用ＥｃｏＲＩ、ＰｓｔＩ将已分离和克隆的马立克氏病病毒糖蛋白Ｄ（ｇＤ）基因从重组ｐＭｇＤ１８质粒中切出，克隆进反转录病毒质粒载体（ＲＣＡＳ）的连接质粒载体ｐＵＣＣｌａ１１２Ｎ的相同位点。用ＣｌａＩ再将ｇＤ重组ｐＵＣＣｌａ１１２Ｎ中的ｇＤ基因切出，构建于ＲＣＡＳ的ＣｌａＩ位点。通过原位杂交筛选重组ＲＣＡＳ，并结合酶切分析鉴定出ｇＤ插入方向正确的重组ＲＣＡＳ。用磷酸钙沉淀法将ｇＤ重组ＲＣＡＳ转染鸡胚成纤维细胞（ＣＥＦ），转染后第９天收集细胞上清，用其感染原代ＣＥＦ，在感染后第４天和第６天收集ＣＥＦ。提取ＣＥＦ基因组ＤＮＡ，用Ｄｉｇｏｘｉｇｅｎｉｎ（Ｄｉｇ）标记ｇＤ基因片段作为探针，通过Ｄｏｔ和Ｓｏｕｔｈｅｒｎｂｌｏｔ检测基因转移情况。用上述上清再次感染原代ＣＥＦ，在第６天用异硫氰酸胍－氯化铯离心法提取感染细胞总ＲＮＡ，用Ｄｉｇ和３２Ｐ标记ｇＤＤＮＡ为探针，分别用Ｄｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ检测ＭＤＶｇＤｍＲＮＡ。结果表明，转染的重组ＲＣＡＳ不仅在细胞内成熟为具传染性的完整病毒。

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。

传染性法氏囊病病毒变异E株感染法氏囊培养细胞凋亡的研究

研究了传染性法氏囊病病毒 (IBDV)变异 E株人工感染体外培养法氏囊细胞的凋亡。电镜观察和DNA电泳分析表明 ,IBDV感染后 4～ 2 4 h,法氏囊培养细胞呈现典型细胞凋亡的形态学特征和生化特征。经流式细胞计检测、荧光染色观察和统计学分析表明 ,IBDV感染后 4～ 2 4 h,法氏囊培养细胞凋亡数量显著增加 (P<0 .0 5,P<0 .0 1)。试验结果说明 IBDV变异

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法。豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验。豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平。统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极显著的相关性(R=0.846,P<0.01);经回归分析,建立了应用豚鼠测定的PD50预测牛口蹄疫Asia-1型灭活疫苗效力的线性回归模型(y=1.625x-0.704;x表示豚鼠PD50结果;y表示牛PD50结果)。F检验(F=15.058,P=0.008)和t检验(t=3.880,P=0.008)证明该线性方程成立。豚鼠的Asia-1型抗体水平随免疫剂量的增加而升高,攻毒时的抗体水平与攻毒后的保护力存在正相关关系。本研究初步证明,在未来的效力检验中豚鼠可作为试验动物替代牛进行口蹄疫Asia-1型灭活疫苗效力检验。