B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies

In this study,the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160(epitope1),tandem repeat 200-213(epitope2(+2)) and the combination of two epitopes(epitope1-2)was genetically cloned into the prokaryotic expression vector pPROExHTb and pGEX4T-1,respectively.VP1 and the fused epitopes GST-E1,GST-E2(+2)and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrat...     

DNA vaccination against foot-and-mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity

BACKGROUND: Foot-and-mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge. MATERIALS AND METHODS: In this study, mice were immunized via electroporation with four cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. RESULTS: Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all four cDNAs that we tested. CONCLUSIONS: These results suggest that the antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward Th2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge.     

同源重组法制备口蹄疫病毒多基因重组腺病毒

通过细菌内同源重组的方法成功构建了含有O型口蹄疫病毒P1—2A和3C蛋白酶基因和3D基因的重组腺病毒表达载体。首先将P1—2A、3C和3D基因亚克隆连接到穿梭质粒pShuttle—CMV上,再将重组穿梭质粒用PmeI线性化后电转化携带有腺病毒骨架载体pAdeasy—1的大肠杆菌BJ5183感受态菌,经细菌内同源重组产生pAdcmv—p12x3c和pAdcmy—p12x3cd重组腺病毒质粒,经序列测定证实目的基因已正确的插入到腺病毒骨架载体中。重组腺病毒质粒经PacI线性化后转染HEK293细胞,转染1w内细胞出现典型病变。取转染细胞裂解液上清连续传代至第4代时,细胞于24～48h内即病变完全,收取接毒后24h细胞进行。PCR和RT—PCR检测,表明目的基因已整合到腺病毒基因组内,且在mRNA水平上有表达。取第4、6、8和10代病毒,用蛋白酶K处理后可扩增出目的基因,证明此重组病毒可稳定存在。本研究为FMDV腺病毒活载体疫苗的研究奠定了基础。     

共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

[目的]筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞(Madin-Darby bovinekidney,MDBK)株.[方法]采用聚合酶链式反应(Polymerase chain reaction,PCR)方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因,将两基因依次插入逆转录病毒载体pBABE-puro.重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞.产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞.利用克隆环套取法得到单克隆细胞.经间接免疫荧光和酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay,ELISA)方法检测MDBK细胞中衣壳蛋白的表达,并在电镜下观察口蹄疫病毒空衣壳.[结果]成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株,衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳.[结论]该研究为口蹄疫亚单位疫苗的研制提供了实验材料.     

口蹄疫病毒株China／99L区段核苷酸序列测定及比较分析

以口蹄疫病毒株China/ 99RNA为模板 ,反转录并扩增目的cDNA ,然后与 pGEM TEasy载体连接并转化JM10 9菌株 ,提取的重组质粒用电泳、PCRampos和EcoR1酶切法鉴定。该毒株与A10、O1K、O1Campos和TW 45毒株的核苷酸序列差异率分别为 15 .2 7%、15 .5 6 %、15 .5 6 %和 15 .49% ;氨基酸序列差异率分别为 8.2 9%、8.76 %、9.2 2 %和 10 .14%。五个毒株的L/P1连接处均为苷氨酸 (Gly) /异亮氨酸 (Ile)。序列比较表明 ,T C、A G和A C转换率较高 ,是点突变的热点核苷酸 ,是影响氨基酸稳定的因素之一。第 43 5 3、95 10 5、10 8 111、146 15 3、16 1 173、183 188和 182 187区域极有可能是L蛋白酶的活性中心 ,第 48位的H、5 1的C、6 5位的E、95位的H、10 9位的H、138位的H、148位的H和 16 5位的E可能是L蛋白酶的活性位点 ,它们在维持蛋白质的空间构像和功能方面具有重要作用     

Synthetic Peptide-based Vaccine and Diagnostic System for Effective Control of FMD

We have designed synthetic peptides corresponding to two different regions of the genome of foot-and-mouth disease virus (FMDV) that are effective as (a) a vaccine or (b) a diagnostic reagent which differentiates convalescent from vaccinated animals, respectively. The peptide vaccine is based on a sequence from the prominent G-H loop of VP1, one of the four capsid proteins. The sequence was optimized by the inclusion of a cyclic constraint and adjoining sequences, and broader immunogenicity was obtained by the incorporation of consensus residues at hypervariable positions. The peptide also included a promiscuous T-helper epitope for effective immunogenicity in outbred populations of large animals.The diagnostic reagent, a peptide based on non-structural (NS) protein 3B, is used in immuno-assays for the detection of antibodies. Antibodies to this NS protein are present in the sera of infected animals but not in the sera of vaccinated animals. The VP1 peptide can be used in complementary immuno-assays for confirmation of NS test results and to monitor for vaccination. This system for differential diagnosis is important to establish the disease-free status of a country.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

Induction of a protective antibody response to FMDV in mice following oral immunization with transgenic <Emphasis Type="Italic">Stylosanthes</Emphasis> spp. as a feedstuff additive

The expression of antigens in transgenic plants has increasingly been used as an alternative to the classical methodologies for the development of experimental vaccines, and it remains one of the real challenges in this field to use transgenic plant-based vaccines effectively as feedstuff additives. We report herein the development of a new oral immunization system for foot and mouth disease with the structural protein VP1 of the foot and mouth disease virus (FMDV) produced in transgenic Stylosanthes guianensis cv. Reyan II. The transgenic plantlets were identified by polymerase chain reaction (PCR), Southern blotting, and northern blotting; and the production of VP1 protein in transgenic plants was confirmed and quantified by western blotting and enzyme-linked immunosorbent assays (ELISA). Six transformed lines were obtained, and the level of the expressed protein was 0.1–0.5% total soluble protein (TSP). Mice that were orally immunized using studded feedstuff mixed with desiccated powder of the transgenic plants developed a virus-specific immune response to the structural VP1 and intact FMDV particles. To our knowledge, this is the first report of transgenic plants expressing the antigen protein of FMDV as feedstuff additives that has demonstrated the induction of a protective systemic antibody response in animals. These results support the feasibility of producing edible vaccines from transgenic forage plants, and provide proof of the possibility of using plant-based vaccines as feedstuff additives.     

口蹄疫病毒株AF72 VP3的结构模拟与分析

以口蹄疫病毒株AF72 RNA为模板,反转录并扩增目的基因,PCR纯化产物与pGEM TEasy载体连接并转化JM109菌株,用凝胶电泳、PCR和Spe I/Sph I双酶切法鉴定为阳性的重组质粒进行测序.比对测序结果确定AF72 VP3的核苷酸序列,利用同源建模的方法建立AF72 VP3结构蛋白的3D结构,在此基础上,综合亲水性、可塑性、抗原指数以及表面可能性等参数预测AF72 VP3结构蛋白的B细胞抗原表位.分析表明,口蹄疫病毒VP1、VP2、VP3和VP4在核苷酸水平上的变异率是无差异的(P＞0.05);而它们在氨基酸水平上的变异率差异显著(P＜0.05).该毒株与20株源于GenBank中的VP3氨基酸序列比对发现其保守区主要位于第1～24、24～35、36～42、45～56、65～122、124～172、177～210、211～219位.AF72 VP3结构蛋白三维空间结构可分为A、B和C 3个结构区域,蛋白呈现较规则的空间构象,其中18～23、30～44、60～75、113～124、130～142、193～220氨基酸区段是AF72VP3结构蛋白可能的B细胞抗原表位区域,该结果将为进一步的FMDV多表位疫苗研究提供更有价值的参考信息.