Asia 1型口蹄疫病毒前导蛋白（L^pro）亚细胞定位

为研究Asia 1型口蹄疫病毒（FMDV）前导蛋白（Lpro）亚细胞定位,利用RT-PCR方法获得L基因,将其定向克隆入pEGFP-N1真核表达载体,经PCR扩增、酶切鉴定及序列测定分析,将鉴定为阳性重组表达质粒命名为pEGFP-L。利用脂质体介导法将pEGFP-L转染BHK-21细胞,用荧光显微镜观察和Western blotting方法检测目的基因的表达,经碘化丙啶（PI）染色后在激光共聚焦显微镜下观察Lpro的亚细胞定位。结果表明,成功构建重组表达质粒pEGFP-L;FMDV L基因在BHK-21细胞中得到表达;Western blotting证实表达的Lpro具有反应活性;激光共聚焦显微镜观察发现Lpro在BHK-21细胞中呈弥散性分布。     

AsiaⅠ型口蹄疫病毒VP1基因结构预测及亚细胞定位

利用PCR方法扩增到VP1基因,序列测定后利用Mega4．0、swiss—model、GOR4和RasMol软件预测了VP1基因的二、三级结构。将VP1基因融合EGFP基因后定向克隆入PcDNA3．1（＋）真核表达载体,构建正确的重组质粒命名为PVP1E,在脂质体介导下将PVP1E质粒转染BHK-21细胞,WesternBlotting试验证实VP1基因成功表达,经DAPI细胞核染色后在共聚焦显微镜下观察VP1亚细胞定位。结果表明本研究预测了VP1基因的二、三级结构,其在BHK-21细胞中呈现以细胞核为主的弥散性分布,VP1基因亚细胞定位及结构预测为进一步深入探究AsiaI型FMDVVP1结构和功能提供丰富的资料。     

口蹄疫病毒前导蛋白的分子生物学研究进展

口蹄疫病毒（Foot-and-mouth disease virus ,FMDV）编码的前导蛋白,不仅是一个重要的蛋白酶,而且对于病毒来说是一个重要的毒力因子,其能够作用于宿主细胞的特异性蛋白,抵抗宿主细胞所产生的抗病毒效应。本文章简述了口蹄疫病毒前导蛋白酶的基本特性,例如前导蛋白的基因结构与病毒复制的关系,前导蛋白酶活性的具体特点,以及前导蛋白影响病毒毒力的分子机制。     

禽流感病毒NS1蛋白亚细胞定位研究

为深入研究NS1蛋白的生物学功能,了解NS1蛋白在哺乳动物细胞中的分布和亚细胞定位情况.采用RT-PCR扩增出禽流感病毒NS1基因片段,克隆至载体pEGFP-N1,经酶切和测序鉴定后,将构建的重组质粒pEGFP-N1-NS1经脂质体介导转染293T细胞和Hela细胞,在荧光显微镜和激光扫描共聚焦显微镜下观察NS1在细胞...     

抗Asia1型口蹄疫病毒单克隆抗体的制备及抗原捕获ELISA检测口蹄疫病毒的研究

本研究用纯化的Asial型口蹄疫病毒(Foot and mouth disease virus,FMDV)免疫BALB/c小鼠,按常规单克隆抗体技术方法,经筛选获得6株能稳定分泌抗Asial型FMDV单抗的杂交瘤细胞株。以牛抗口蹄疫病毒IgG为捕获抗体,选择一株单抗(184)用辣根过氧化物酶标记(HRP-184)作为检测抗体,建立了检测Asial型FMDV的抗原捕获ELISA方法。该方法可检出0.5859μg纯化Asial型FMDV抗原和2.5×10~3TCID_(50)病毒,对O型FMDV、牛结核病、牛肺疫、牛流热、赤羽病、牛传染性鼻炎等病毒进行检测,均为阴性,无交叉反应发生。本研究建立的FMDV抗原捕获ELISA方法,具有敏感性高、特异性强和重复性好的特点,可用于Asial型FMDV的特异性检测。     

O型和Asia1型口蹄疫病毒感染RT-PCR鉴别诊断方法的建立

根据GenBank中O型和Asia1型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)的vp3、vp1和2A基因序列,并与其它血清型FMDV的对应基因序列进行比较,设计用于扩增O型和Asia1型FMDV vp1基因的特异性引物,建立O型和Asia1型FMDV RT-PCR鉴别诊断方法。本方法首先用通用型引物进行RT-PCR,确定是否为FMDV感染,然后用特异性引物鉴别O型或Asia1型FMDV的感染。用vp1基因序列分析进行符合性试验,验证了该方法所具有的特异性和敏感性。本方法可用于O型和Asia1型FMD的快速诊断及流行病学调查。     

AsiaⅠ型口蹄疫病毒VP1基因在昆虫细胞/杆状病毒中的表达

利用杆状病毒表达系统对Asia Ⅰ型口蹄疫病毒(foot-and-mouth disease virus,FMDV)VPl基因在Sf9昆虫细胞中进行表达,为研究Asia Ⅰ型FMDV VP1蛋白功能及建立Asia Ⅰ型FMDV血清学诊断方法奠定基础.采用PCR方法从pGEM-T-Easy-Asia Ⅰ型VP1质粒中扩增VP1基因,将其插入杆状病毒转座载体pFastBacHTA,构建的重组质粒pFast-BaeHTA-VPl再转入DH10Bae感受态细胞,经三重抗性与蓝白斑筛选,获得杆状病毒重组质粒Baemid-VPl,然后转染Sf9昆虫细胞.PCR鉴定证实VP1基因正确地插入到Bacmid中,成功构建了杆状病毒重组质粒Baemid-VP1,SDS-PAGE和West-ern-blotting检测结果表明,VP1基因在Sf9昆虫细胞中表达出约26.5 ku的VP1蛋白.将可溶性表达的融合蛋白用Ni-NTA亲和层析方法进行纯化,通过ELISA分析,能特异性地检测出Asia Ⅰ型口蹄疫病毒阳性血清.Asia Ⅰ型FMDV VP1基因在杆状病毒表达系统中的成功表达为Asia Ⅰ型FMDV VP1蛋白的抗原性及血清学抗体水平检测研究奠定了基础.     

IPTG诱导浓度、时间对AsiaⅠ型口蹄疫病毒VP1蛋白表达的影响

将口蹄疫病毒VP1基因插入原核表达载体pET-28a中,转化至大肠杆菌BL21.用不同的IPTG浓度与不同诱导时间诱导菌株表达VP1蛋向,分析不同IPTG浓度及不同诱导时间对蛋白表达量的影响,以确定最佳表达条件.结果表明,在一定范围内,随IPTG浓度的增高,表达量并不会随之增大;而在菌体对数生长期内,随诱导时间的延长,表达量会随之增大.最后确定当IPTG浓度为3 mmol/L,诱导表达到最长时间9 h时,VP1蛋白表达量最大.     

猪流感病毒聚合酶PB1蛋白亚细胞定位的研究

猪流感病毒PB1蛋白在其复制中起着转录作用,是病毒复制所必需的蛋白。克隆了猪流感病毒的PB1基因,构建重组真核表达载体p3xFLAG-CMV-7.1-PB1,利用脂质体转染Vero细胞后,分别用Western blot和IFA检测重组蛋白FLAG-PB1蛋白的表达,结果表明重组蛋白能在真核细胞中得到表达,其亚细胞定位为细胞核表达,与其功能密切相关,为以后流感病毒的相关研究打下了基础。     

口蹄疫病毒前导蛋白酶的研究进展

前导蛋白酶(leader protease,Lpro)是FMDV基因组编码具有酶学活性的病毒产物之一。其可被2个串联的翻译起始密码子进行翻译表达,且以第2个翻译起始密码子进行翻译起始的产物居多。Lpro含有球状区域,碳端有一柔性杆状结构,其执行蛋白酶生物学功能的活性集团的氨基酸序列可能是第144位的Lys、148位的His和163位的Asp。保守区氨基酸残基在维持蛋白的空间结构和功能方面具有重要作用。此种蛋白酶的C端编码序列区中的同义密码子使用模式在病毒蛋白的翻译过程具有重要的生物学意义。Lpro能够剪切病毒编码的多聚蛋白,且使能宿主细胞中特定的蛋白质降解,是FMDV的重要毒力因子。Lpro通过抑制干扰素(IFN)的分泌降低免疫监视系统对FMDV的监视能力,从而逃避感染动物对FMDV侵染的抵抗力。     

一株Asia 1型口蹄疫病毒的全基因组序列测定及比较分析

参考GenBank上已发表的口蹄疫病毒（FMDV）全长基因组序列,设计了覆盖基因组全长的数对引物,通过RT-PCR方法对1株Asia1型（简称As01）FMDV进行分段克隆及序列测定,将测序结果利用软件进行拼接。结果表明,该毒株的FMDV基因组[不合poly（C）]全长8180nt,其中编码区为6987nt,5’和3’非编码区（UTR）分别为1078nt和95nt,3＇UTR之后为20nt的poly（A）尾巴。将As01株与其他FMDV参考毒株利用分子生物学软件进行多序列比对分析表明,As01株与Asia 1／Jiangsu／China／2005、Asia 1／Isr 1／3／63、ZB／CHA／58等Asia 1型FMDV遗传进化关系较为密切,而与O型和A型FMDV遗传关系较远。     

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。     

Sequence variability in the structural protein-encoding region of foot-and-mouth disease virus serotype Asia1 field isolates

A total of 30 field isolates of foot-and-mouth disease virus (FMDV) serotype Asia1 belonging to two different lineages and five isolates belonging to a divergent group as delineated earlier in 1D (encodingVP1 protein) gene-based phylogeny were sequenced in the structural protein (P1) coding region. Phylogenetic comparison of these isolates along with some of the published exotic sequences revealed the presence of five different lineages around the world. Similar grouping pattern was observed for the P1 region and 1D gene-based phylogeny, where the Indian isolates were clustered in two genetic lineages. The recently identified divergent group of virus falls into a separate sub-cluster. Similar grouping was also observed in L gene-based phylogeny. Comparison of amino acid sequences identified lineage-specific signature residues in all the structural proteins. Comparison of Asia1 field isolates at the identified key residues of other FMD viruses involved in the formation of the heparan sulfate-binding ligand confirmed many of them to be conserved and the presence of VP3(56) Arg suggested their cell culture adaptation. Although a considerable genetic variation was observed among the isolates of present study, all of them tested in micro-neutralization test were serologically related to the vaccine strain.     

Antigenic characterization of foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and monoclonal antibodies

Foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates (n = 100) were compared using a panel of 11 monoclonal antibodies (Mab) in sandwich ELISA. The majority (over 89%) of the isolates showed either homologous (76% and above reactivity) or reduced affinity (20-75% reactivity) for the Mabs 2A, 13, 40, 34 and 81, suggesting that these Mab binding epitopes are conserved, whereas a more variable reactivity was observed for the Mabs B3, 1A, 24, 72, 82 and 89. Polyclonal relationship ('r' value) of the field isolates in liquid phase blocking (LPB) ELISA was examined, and the mean 'r' value was 0.62 relative to vaccine virus IND 63/72. Some of the field isolates (n = 34) were tested in virus neutralization test (VNT) and showed an 'r' value of >0.40. Although a minor antigenic difference was observed in the Mab profiling study, there has not been large antigenic divergence between reference virus and field viruses, thereby providing evidence of wide antigenic coverage of the vaccine strain.     

Screening and identification of B cell epitopes of structural proteins of foot-and-mouth disease virus serotype Asia1

The objective of this study was to screen and identify the B cell epitopes of structural proteins of foot-and-mouth disease virus (FMDV) serotype Asia1. The complete amino acid sequence of all the four structural proteins (P1 region) was analyzed using the DNAStar Protean system. Seventeen peptides were predicted and selected as potential B cell epitopes. The potential B cell epitope genes were cloned into the pGEX-6P-1 plasmid, then expressed and purified. The resulting 17 glutathione S-transferase (GST) fusion peptides were detected by Western blot and ELISA for evaluation of their antigenicity. Six of the 17 fusion peptides were identified successfully by sera from rabbits immunized with the purified P1 polyprotein of FMDV type Asia1. The six fusion proteins were epi1-1 (VP1:₁TTTTGESADPVT₁₂), epi1-2 (VP1:₁₇NYGGETQTARRLH₂₉), epi1-6 (VP1:₁₉₄TTQDRRKQEIIAPEKQTL₂₁₁), epi2-2 (VP2:₄₀EDAVSGPNTSG₅₀), epi3-1 (VP3:₂₆YGKVSNPPRTSFPG₃₉), and epi4-2 (VP4:₃₀YQNSMDTQLGDN₄₁). The results of this study lay a foundation for further study of the structure and function of the structural proteins and may aid in the design of an epitope vaccine against foot-and-mouth disease (FMD) type Asia1. This study has also shown that the bioinformatics method, in combination with molecular biology methods can be used to map the B cell epitopes on viral proteins.     

表达A型口蹄疫病毒衣壳蛋白重组腺病毒的构建

为构建表达A型口蹄疫病毒(FMDV)衣壳蛋白的重组腺病毒,本研究通过人工合成A型FMDVP1-2A、2B和3C融合基因,将其克隆到腺病毒穿梭载体pShuttle-CMV中,利用E.coli BJ5183内同源重组将目的基因插入腺病毒骨架质粒pAdEasy-1中,获得携带A型FMDV P1-2A-2B-3C基因的重组AdEasy-1。该重组质粒经PacⅠ线性化后转染AD-293细胞,获得重组腺病毒rAd-A09。经PCR检测,该重组腺病毒在传代过程中目的基因稳定存在,病毒滴度在第8代时可达到108.5TCID50/mL。间接免疫荧光检测和western blot分析表明,rAd-A09在AD-293细胞中产生FMDV的结构蛋白VP0、VP1和VP3。该重组腺病毒的构建为口蹄疫新型疫苗的研究奠定了基础。     

Kinetics of immune response to foot-and-mouth disease virus (type Asia 1) in experimental cattle

Humoral and mucosal (secretory antibody)immune response to FMDV type Asia 1 in cattle was analyzed after vaccination and infection using virus neutralizing test (VNT). Vaccination (1/16th the usual dose) failed to protect cattle from generalized clinical disease following experimental FMDV Asia 1 infection. Our results showed that infection induced higher and prolonged serum antibody titres indicating antigen mass is important for optimal immune response. Experimental FMDV infection induced significant secretory antibody (mucosal) response in cattle. Though, there was no difference in the serum antibody response between the cattle that developed generalized infection (unprotected) and those with only localized infection (protected), secretory antibody response differed, wherein the unprotected cattle had higher secretory response than protected cattle. Thus, FMDV Asia 1 infection stimulates a similar serum antibody response and a unique secretory antibody response among the infected cattle. An erratum to this article can be found at     

Emergence of a novel subgroup within the widely circulating lineage of foot-and-mouth disease virus serotype Asia 1 in India

The complete VP1 encoding (1D) gene of 54 foot-and-mouth disease (FMD) virus serotype Asia1 field isolates, most of which were isolated during 2000 and 2001, was sequenced. The phylogenetic analysis identified a novel subgroup (>10% nucleotide divergence) within the widely circulating lineage of this serotype. The newly emerged viruses were responsible for disease outbreaks in both cattle and buffaloes and were present in six different states in the country. Amino acid sequence comparison of these isolates revealed significant sequence divergence at many of the amino acid positions in comparison to those of lineage VI-A and C. Emergence of such viruses may affect the efficacy of vaccine strain currently used for protection against FMD in India.