cDNA-cloning and expression of VP1-specific sequences of foot-and-mouth disease virus types A5 and O1

The RNA genome of foot- and mouth disease virus strains A5 Westerwald and O1 Lausanne has been reverse-transcribed and cloned in lambdaphages or plasmids. Identification of cDNA-clones containing VP1-specific sequences was achieved by hybridization, restriction mapping, and sequence analysis. VP1-coding cDNA-fragments were subcloned into the expression vector pEX which led to synthesis of fusion proteins with beta-galactosidase. These fusion proteins reacted with anti-VP1 antibodies on a Western blot, but were not capable of inducing neutralizing antibodies to mice. This seemed to suggest a tertiary structure of the VP1-epitopes unlike those of native VP1. Other attempts are discussed to construct VP1-fusion proteins folding similarly to the native viral protein structure.     

口蹄疫病毒OH株结构蛋白基因VP1的克隆与表达

采用RT—PCR方法扩增获得了O型口蹄疫病毒的主要免疫原VP1基因，将其插入pMDl8-T载体进行序列分析，结果表明，所获得的基因片段含有完整的FMDV结构蛋白VP1编码区。根据表达载体pQE-Trisystem的克隆位点序列和该VPl基因片段的末端序列设计了1对表达引物，以重组pMD-T—VP1阳性质粒为模板，扩增获得了VP1基因，通过酶切将其克隆至表达载体pQE—Trisystem上。经测序证实，重组表达质粒所含的外源基因VP1编码框正确无误。将重组表达质粒pQE—VP1转化至大肠埃希氏菌M15，通过IPTG诱导促使VP1基因高效表达，SDS—PAGE和Western—blot分析表明，表达产物大小与预期的结果（26ku）一致，且具有良好的反应原性。以2mmol／LIPTG诱导表达5h时表达量最高，其中70％～80％的目的蛋白存在于菌体裂解后的上清中，表明外源基因VP1主要以可溶性方式表达。     

6株猪O型口蹄疫病毒VP1基因的克隆与序列分析

根据口蹄疫病毒（FMDV）VP1基因的序列，设计并合成了2对用于扩增VP1基因的引物。从组织中提取总RNA，首先用P1、P2引物对6株猪。型口蹄疫病毒进行RT—PCR扩增，获得1000bp的片段；再用P3、P4引物进行巢式PCR扩增，结果获得850bp的片段。将850bp的片段克隆到pMD18—-T载体中，通过PCR鉴定，将阳性重组质粒进行测序并分析。结果发现6株FMDV的核苷酸同源性为80．2％～99．4％，其推导的氨基酸序列同源性为86．9％～99．5％；构建遗传发生树，发现6株FMDV属于两个不同的基因型，其中的Shunde00、Sihui01、Shenzhen99、Fushan01株属一个基因型（与Hongkong93、广东86分离株属同一基因型）；Guangzhou99、Shenzhen00株属另一个基因型（与UKG-12—2001株、JPN2000株属同一基因型）。通过对口蹄疫病毒VP1基因的测序与分析，了解其变异情况，为科学地防控FMD提供分子水平的依据。     

Asia 1型口蹄疫病毒VP1基因的表达鉴定及其多抗制备

本研究旨在表达口蹄疫病毒(FMDV)的VP1全基因并制备特异性的多克隆抗体。利用PCR方法扩增Asia 1 IND 49197株VP1全基因,将其克隆至原核表达载体pET-30a(+)中,在大肠杆菌BL21中进行表达。SDS-PAGE结果显示表达产物分子量约为31.6ku,以包涵体的形式存在。通过Ni-NTA Purification System纯化后进行western blot和间接ELISA分析,结果显示重组蛋白能够被FMD阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,ELISA测定抗体效价为1∶20480,病毒中和试验测定抗体效价为1∶64。本研究所表达的VP1蛋白可用于开发检测Asia1口蹄疫抗体的诊断试剂,所制备的多克隆抗体为进一步研究VP1的结构、功能以及抗原表位的鉴定提供了条件。     

口蹄疫病毒VP2基因的原核表达及抗原性检测

将口蹄疫病毒VP2基因连接到原核表达载体pPROEX^TM HTb上，获得重组质粒pPR-VP2。将此质粒转化感受态菌DH5α中，经终浓度为1．0mmol／L的IPTG诱导，1h～6h依次收集菌液，SDS-PAGE电泳分析，在4h后表达量可达到高峰。经过软件分析，表达产物占总菌体蛋白的23．1％，大小为33Ku左右。根据Ni-NTA纯化系统说明操作，获得较纯的VP2融合蛋白。以牛抗O型口蹄疫病毒血清为第一抗体，通过Western blot和Dot ELISA鉴定，纯化后的VP2融合蛋白可以与牛抗O型口蹄疫病毒血清发生特异性反应。     

Sequence and phylogenetic analysis of the non-structural 3A and 3B protein-coding regions of foot-and-mouth disease virus subtype A Iran 05

The A Iran 05 foot-and-mouth disease virus (FMDV) subtype was detected in Iran during 2005 and has proven to be highly virulent. This study was undertaken to focus on molecular and phylogenetic analysis of 3A and 3B coding-regions in the A Iran 05 field isolate. To assess the genetic relatedness of A Iran 05 isolate the nucleotide and predicted amino acid sequences of the 3AB region of type A FMDV isolates were compared with twenty previously described type A FMDV isolates. The phylogenetic tree based on the 672 bp 3AB gene sequences of type A FMDV from thirteen different locations clustered them into five distinct lineages. The A Iran 05 isolate clustered in lineage A along with four type A variants and was closely matched with viruses isolated in Turkey and Pakistan during 2005~2006. The number of protein sequence differences exhibited by each of the isolates revealed that A Iran 05 isolate contains three amino acid substitutions at positions 47 and 119 of 3A and 27 of the 3B coding region. The nucleotide identity between A Iran 05 and the other four isolates of lineage A was estimated to be 98%.     

Sequence analysis of the protein-coding regions of foot-and-mouth disease virus O/HK/2001

The nucleotide sequence of the protein-coding region of foot-mouth-disease virus (FMDV) strain O/HK/2001 was determined and compared with the sequences of other FMDVs that were registered in GenBank. The protein-coding region was 6966 nucleotides in length and encoded a protein of 2322 amino acid residues. Comparison of the nucleotide sequence and its deduced amino acid sequence with those of other isolates indicated that O/HK/2001 belonged to the Cathay topotype. A genomic coding region nucleotide sequence phylogenetic tree of several FMDV-O isolates showed that O/HK/2001 was most closely related to FMDV isolates found in Taiwan during 1997, and especially shared significant similarity to HKN/2002, suggesting that the virus causing outbreaks in Hong Kong was genetically most-closely related to that causing an outbreak of type O in Taiwan. Mutations in O/HK/2001 were revealed, including frequent substitutions in the VP1 and L proteins, and deletions involving 10 amino acid residues in the 3A protein. This study was undertaken to assess the regional variation of prevalent FMDV type O viruses and to establish a sequence database for FMDV molecular epidemiological investigation.     

C型口蹄疫病毒VP1基因的原核表达及重组蛋白的纯化

利用PCR技术扩增获得C型口蹄疫病毒VP1基因,将其克隆到原核表达载体pET-30α(+)上,构建重组表达质粒pET-VP1。质粒pET-VP1转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导VP1基因的表达,收集不同时间的菌液进行SDS-PAGE、Western blot分析,结果得到分子量约为33 ku的目的条带,表达产物占菌体总蛋白的35%,且该目的条带能被C型FMDV阳性血清识别,说明VP1基因在大肠杆菌中得到高效表达。融合表达蛋白经镍柱纯化,重组蛋白纯度达90%以上。     

牛口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法的建立

根据牛口蹄疫O型3个拓扑型VP1序列设计3条合成肽,以此为包被抗原建立牛口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法,并对该方法敏感性、特异性和重复性进行验证。结果显示,该方法敏感性为96.7%,特异性为99.1%,批内和批间变异系数分别为2.1%~9.8%和3.5%~10.7%。与2种商品化试剂盒比较,符合率分别为93.5%和85.9%。结果表明,该方法敏感性好、特异性强、稳定性高,可用于牛口蹄疫O型抗体水平检测。     

鸽源新城疫病毒的鉴定及HN基因遗传演化分析

根据发病鸽群的临床症状、病理剖检、组织病理学变化,并结合RT-PCR方法对武汉地区某鸽场疑似鸽新城疫进行了确诊,同时对分离的2株鸽源新城疫病毒(NDV)进行了HN基因结构特征及其与已知毒株的遗传相关性分析。结果表明:两株鸽源NDV分离株HN基因片段均含有6个保守的糖基化位点。2株NDV分离株HN基因序列与参考株同源性在80.0%~97.2%,与湖北株(AY225110.HB92)、印度株(KM056357.ndv61/B1)、江苏株(GQ245832.YC-24-07-CH)、陕西株(JF795531.HX01)在同一分支上,为基因II型。研究为武汉地区鸽新城疫的防控提供了理论依据。     

Molecular epidemiological studies on foot-and-mouth disease type O Taiwan viruses from the 1997 epidemic

Sequence diversity was assessed of the complete VP1 gene directly amplified from 49 clinical specimens during an explosive foot-and-mouth disease (FMD) outbreak in Taiwan. Type O Taiwan FMD viruses are genetically highly homogenous, as seen by the minute divergence of 0.2-0.9% revealed in 20 variants. The O/HCP-0314/TW/97 and O/TCP-022/TW/97 viral variants dominated FMD outbreaks and were prevalent in most affected pig-raising areas. Comparison of deduced amino acid sequences around the main neutralizable antigenic sites on the VP1 polypeptide showed no significant antigenic variation. However, the O/CHP-158/TW/97 variant had an alternative critical residue at position 43 in antigenic site 3, which may be due to selective pressure in the field. Two vaccine production strains (O1/Manisa/Turkey/69 and O1/Campos/Brazil/71) probably provide partial heterologous protection of swine against O Taiwan viruses. The type O Taiwan variants clustered in sublineage A1 of four main lineages in the phylogenetic tree. The O/Hong Kong/9/94 and O/1685/Moscow/Russia/95 viruses in sublineage A2 are closely related to the O Taiwan variants. The causative agent for the 1997 epidemic presumably originated from a single common source of type O FMD viruses prevalent in neighboring areas.     

貂阿留申病病毒结构蛋白VP1基因分子特征分析

目的研究貂阿留申病病毒(ADV)结构蛋白VP1基因分子空间结构特征,探讨貂阿留申病病毒致病机制。方法根据Genbank公布的全基因序列设计三对引物,PCR扩增并克隆到pMD-18T载体,阳性重组质粒鉴定、测序验证,拼接后进行生物信息学分析。结果获得了全长2064bpVP1基因,编码688个氨基酸;与细小病毒属中PPV-Nanjing200801相似性最大(51.2%);遗传进化树显示该基因编码蛋白与细小病毒属VP1亲缘关系最近。该蛋白是一种保守不含信号肽的外膜蛋白,具有7个潜在的N-糖基化位点和34个磷酸化位点。二级结构分析显示无规则卷曲含量最高,达68.75%,α螺旋、β折叠分别为16.42%和14.83%;同源建模比对,构建了具有较高合理性和可靠性的三维空间结构。结论预测抗原表位主要位于肽链第254~265、96~112、317~348、514~523、629~645位区段,可为今后开展基因工程疫苗研究奠定基础。     

口蹄疫病毒结构蛋白基因vp1的表达与应用研究

体外克隆口蹄疫病毒vp1基因，构建重组表达载体pET28a-vp1。将此重组质粒转化到受体菌BL21（DE3）中，进行诱导表达，SDS-PAGE和蛋白质印迹分析表明，诱导5h后表达量达到最高，表达产物大小约为33Ku-40Ku，表达蛋白能与口蹄疫病毒阳性血清产生特异性免疫反应。经HPLC纯化后，以重组蛋白为抗原，建立检测VP1蛋白抗体的ELISA方法，检测猪牛血清样品，免疫抗体检测结果与口蹄疫液相阻断ELISA检测结果呈正相关，能反映出免疫抗体动态变化，对临床样品口蹄疫病毒血清抗体检测，两种方法有一定相关性，但不显著。所以以重组VP1蛋白为检测抗原的ELISA方法有望用于口蹄疫免疫抗体监测。     

猪口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法的建立

根据猪口蹄疫O型流行毒株VP1序列设计出5条合成肽,以固相法合成并以此合成肽作为包被抗原建立猪口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法,并对该方法敏感性、特异性、重复性等进行验证。结果表明:该检测方法敏感性为96.0%,特异性为99.1%,批内与批间重复试验变异系数小于10.0%。比对试验结果显示,该检测方法与UBI猪口蹄疫病毒VP1结构蛋白抗体酶联免疫吸附试验诊断试剂盒符合率为93.2%,与中国农业科学院兰州兽医研究所口蹄疫O型液相阻断ELISA抗体检测试剂盒符合率为85.7%。该猪口蹄疫O型合成肽VP1结构蛋白ELISA抗体检测方法敏感性好、特异性强、稳定性高、操作简便,可用于检测O型口蹄疫抗体水平。     

Investigation of the influence of peptide-carrier conjugation on the immunological activity of VP1-peptides of foot-and-mouth disease virus O1-Kaufbeuren

We found coupling of short sequences of VP1 to keyhole limpet hemocyanin (KLH) by means of glutaraldehyde to be a very complex phenomenon which could only be controlled by strict standardization of components and reaction conditions. Considering the results, we may conclude that big immunogenic proteins, like KLH, are advantageous for achieving sufficient and specific antibody response with neutralizing activity. When using KLH, we did not find simple dependence of immunogenicity or neutralizing activity on the incorporation rate of hapten in KLH in a region between 50 and 200. To develop a synthetic vaccine of good economy, investigations have to be continued, primary with a view to lowering the doses involved.     

O型FMDV VP1基因的真核表达及其产物的生物学活性

根据已克隆的O型口蹄疫病毒VP1基因序列，设计了1对带有Sac Ⅰ和HindⅢ酶切位点的引物，用其将pMD18-T-VP1质粒中的VP1基因亚克隆到真核表达栽体pBlueBacHis2A中，成功地构建了重组表达质粒pBlueBacHis2A—VP1（633bp）。将pBIueBacHis2A—VP1（633bp）质粒与Bac-N—Blue^TM DNA共转染sf9昆虫细胞，蚀班筛选重组病毒后，将纯化的重组病毒感染sf9细胞，获得了32．6ku的目的蛋白条带。Dot—ELISA分析结果表明，该表达产物具有反应活性，可用于建立间接ELISA方法，进行口蹄疫病毒抗体检测。     

A型口蹄疫病毒VP1基因序列的基因分型研究

为设计A型FMDV基因分型探针建立其3个基因型的数据库,以美国国家生物技术信息中心(NCBI)基因库和英国口蹄疫世界参考实验室(FMDWRL)基因库中所登记的血清A型口蹄疫病毒(FMDV)VP1基因序列为研究对象,运用双序列比对和构建系统发育树的方法对未知基因型的VP1序列进行基因分型并比较两种方法的分型结果。结果表明,两种分型方法的分型率均达到92%,分型结果基本一致。运用这两种方法都可实现对A型FMDV VP1序列的基因分型。     

口蹄疫病毒VP2基因的真核表达及其产物抗原性的检测

利用PCR技术，扩增出口蹄疫病毒（FMDV）VP2基因，并克隆到杆状病毒转移载体pBlueBacHis2A上。用重组质粒pB-VP2与重组病毒同时转染sf9昆虫细胞，获得了重组病毒。经过蚀斑筛选纯化后，感染sf9细胞，表达VP2融合蛋白，分子质量为33ku左右。以牛抗O型FMDV血清为第一抗体，通过Western-blotting和Dot-ELISA鉴定，说明VP2基因在真核表达系统中获得正确表达，且可以与牛抗。型FMDV血清发生特异性反应。     

鸭甲肝病毒VP1基因的克隆和序列分析

鸭病毒性肝炎的致病病原包括鸭肝炎病毒(DHV)和鸭星状病毒(DAstV)[1-2].DHV可分为血清1型[3-5]、台湾新型[6]和韩国新型[7],分别对应于基因A型、B型和C型[8].DAstV指传统的DHV血清2型(DHV-2)[9-11]和3型(DHV-3)[12] (Knowles,个人通讯),其中DHV-2已更名为DAstV-1[2].为避免混淆,已建议将鸭肝炎病毒改称为鸭甲肝病毒(duck hepatitis A virus,DHAV)[13],而DHV-3也应更名为DAstV-2.     

Identification of a conformational epitope on the VP1 G-H Loop of type Asia1 foot-and-mouth disease virus defined by a protective monoclonal antibody

Although neutralizing antigenic sites of foot-and-mouth disease virus (FMDV) can be defined by selection of monoclonal antibody (MAb) escape mutants, no conformational neutralizing epitope on the major antigenic site located on the G-H loop of type Asia1 FMDV has been precisely mapped. In this study, we generated a potent neutralizing MAb 3E11, which recognized a conformation-dependent epitope and neutralized FMDV Asia1/YS/CHA/05 in vitro. Importantly, a dose of 5.5 NT(50) of the MAb 3E11 completely protected suckling mice from a dose of 10 LD(50) of homologous virus challenge in vivo. Through a 12-mer random peptide phage display, synthetic peptide analysis and constructing a series of FMDV Asia1/YS/CHA/05 mutants using reverse genetic system, we finely mapped the neutralizing epitope as the 12-amino acid peptide (141)SXRGXLXXLXRR(152). These results provide additional insights into the virus-MAb interaction at the amino acid level and may help in the development of an epitope-based Asia1 FMDV vaccine.