A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli

Abstract The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype Cl virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.     

三种大肠杆菌表达的口蹄疫病毒A型多表位蛋白对猪的免疫原性比较

【目的】研制猪口蹄疫病毒(foot-and-mouth disease virus,FMDV)A型多表位蛋白疫苗,为猪FMDV A型的防控提供安全有效的疫苗。【方法】根据前期试验结果及国内外FMDVA型流行病学信息,设计并合成了3种多表位免疫原基因A10、IA10和FA10。在大肠杆菌BL21(DE3)中诱导表达,表达蛋白纯化复性后,制苗免疫猪。分别于免疫前和免疫后14和28d采血分离血清,用液相阻断ELISA(LPB-ELISA)方法检测血清IgG抗体滴度。免疫28d后用FMDV强毒攻毒,以评估免疫保护效果。【结果】SDS-PAGE和Western blotting结果证实A10、IA10和FA10三种蛋白均获得表达,分子量分别为35、57和64 kDa,与预测蛋白大小一致,且能被FMDV感染阳性血清所识别。LPB-ELISA结果表明,A10+201免疫组IgG滴度低于灭活疫苗组,但高于其他免疫组。攻毒后A10+201免疫组和灭活疫苗免疫组全部猪(5/5)获得保护,IA10+201和FA10+201免疫组80%(4/5)猪保护,A10和FA10免疫组只有20%(1/5)猪保护,而PBS+201组所有猪均未保护。【结论】A10+201免疫保护效果较好,可作为候选疫苗进行进一步评价。     

不同粒径口蹄疫病毒样颗粒-ZIF-8复合物诱导小鼠的体液免疫效果分析

为进一步提高口蹄疫(foot-and-mouthdisease,FMD)病毒样颗粒(virus-likeparticles,VLPs)疫苗的免疫效果，本研究采用仿生矿化方法，将Zn2+和2-甲基咪唑按照不同浓度配比制备了不同粒径的FMDV VLPs-沸石咪唑骨架-8 (zeolitic imidazolate framework-8, ZIF-8)复合物，以探究尺寸效应对免疫效果的影响。结果显示，成功制备出3种不同粒径的FMDV VLPs-ZIF-8，粒径分别约为70、100、1 000 nm。细胞毒性和组织病理学试验表明，3种复合物均具有良好的生物安全性。小鼠免疫试验表明，3种复合物均能明显提高中和抗体和特异性抗体水平，并且随着复合物体积的减小，其免疫效果也随之增强。本研究表明，ZIF-8包封FMDV VLPs可显著增强其免疫效果，且具有尺寸依赖性。     

口蹄疫病毒入侵宿主细胞研究进展

口蹄疫病毒(FMDV)是小RNA病毒科,口蹄疫病毒属的典型成员,是一种基因组大约含有8 400个核苷酸的无囊膜单股正链RNA病毒。大量研究发现识别细胞表面受体并侵入细胞是FMDV感染宿主细胞非常重要的环节;对FMDV而言,利用哪种受体就决定了利用哪种內吞路径。近年来在口蹄疫病毒入侵宿主细胞方面进行了大量研究,在一定程度上解释了口蹄疫病毒感染机制方面的问题,为解决实际生产问题提供了重要依据。对前期工作进行阶段性总结,为后期深入研究口蹄疫病毒致病机制和探索更有效的防治措施提供参考。     

L protease from foot and mouth disease virus confers eIF2-independent translation for mRNAs bearing picornavirus IRES

The leader protease (L^pro) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L^pro is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L^pro, significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).     

构建多顺反子表达载体的有效工具——FMDV 2A

近年来肿瘤多基因治疗倍受关注,然而目前缺少一种有效的构建多顺子的工具。传统的构建多顺反子的工具,如IRES等,由于存在结构大,上下游基因表达差异显著等缺陷,严重限制了其应用。FMDV2A,因其具有结构小、剪切效率高等优点为多基因治疗带来了新的希望。主要介绍了FMDV2A的特性、"剪切"活力及其在构建多顺反子载体中的应用。     

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type

In order to develop an anti-FMDV A Type monoclonal antibody (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of the...     

Develope monoclonal antibody against foot-and-mouth disease virus a type

In order to develop an anti-FMDV A Type monoclonal antibo by (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/O myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of these mAbs against A Type FMDV,were examined using indirect ELISA,the result showed that both mAbs reacted with A Type FMDV.These mAbs may be used for further vaccine studies,diagnostic methods,prophylaxis,etiological and immunological research on FMDV.Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease (SVD) or FMDV O,Asial and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 was classified as IgG2b subtype.The mAbs prepared in this study,are specific for detection of FMDV serotype A,and is potentially useful for pen-side diagnosis.     

Comparative studies of the capsid precursor polypeptide P1 and the capsid protein VP1 cDNA vectors for DNA vaccination against foot-and-mouth disease virus

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies. MATERIALS AND METHODS: Here we evaluated comparatively three different viral antigen-encoding DNA sequences, delivered via two physical means (i.e., gene gun delivery into skin and electroporation delivery into muscle), for naked DNA-mediated vaccination in a mouse system. RESULTS: Both methods gave similar results, demonstrating commonality of the observed DNA vaccine effects. Immunization with a cDNA vector expressing the major viral antigen (VP1) alone routinely failed to induce the production of anti-VP1 or neutralizing antibodies in test mice. As a second approach, the plasmid L-VP1 that produces a transgenic membrane-anchored VP1 protein elicited a strong antibody response, but all test mice failed in the FMDV challenge experiment. In contrast, for mice immunized with the viral capsid precursor protein (P1) cDNA expression vector, both neutralizing antibodies and 80-100% protection in test mice were detected. CONCLUSIONS: This strategy of using the whole capsid precursor protein P1 cDNA for vaccination, intentionally without the use of virus-specific protease or other encoding genes for safety reasons, may thus be employed as a relevant experimental system for induction or upgrading of effective neutralizing antibody response, and as a convenient surrogate test system for DNA vaccination studies of FMDV and presumably other viral diseases.