甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物纳米微球作为口蹄疫病毒核酸疫苗递送载体的特性评价

旨在对制备的甘露糖修饰的壳聚糖聚乳酸-羟基乙酸共聚物[poly （D，L-lactide-co-glycolide），PLGA]纳米微球作为口蹄疫病毒（foot-and-mouth disease virus，FMDV）核酸疫苗递送载体进行评价。采用西佛碱反应和元素分析制备具有一定取代度的甘露糖修饰的壳聚糖衍生物（mannose modified chitosan，MCS），然后，经双重乳化挥发法制备得到甘露糖修饰的壳聚糖PLGA纳米微球（MCS-PLGA-NPs）。采用纳米粒径仪检测MCS-PLGA-NPs粒径分布和表面电势（zeta）、扫描电镜考察其形态、琼脂糖凝胶电泳观察其对质粒的吸附和吸附质粒后抵抗核酸酶降解能力、CCK-8法检测MCS-PLGA-NPs的细胞毒性、激光共聚焦观察巨噬细胞对MCS-PLGA-NPs-质粒DNA复合物的摄取、荧光显微镜和Western blot验证MCS-PLGA-NPs加载质粒DNA在细胞中的表达。元素分析结果表明，成功制备了取代度为5%~10%的MCS。纳米粒径测定和扫描电镜结果表明，MCS-PLGA-NPs的zeta为正值、粒径分布均匀且形态规则呈球形。琼脂糖凝胶电泳结果显示，MCS-PLGA-NPs吸附质粒的能力随着其质量的增加而增强并且可以在一定程度上抵抗核酸酶降解质粒DNA。在细胞毒性试验中，不同浓度的MCS-PLGA-NPs与RAW264.7细胞共孵育24 h后，细胞存活率仍在85%以上。在细胞摄取试验中，用激光共聚焦显微镜可以明显观察到质粒DNA结合到纳米微球表面被RAW264.7细胞摄取。荧光显微镜和Western blot试验证明MCS-PLGA-NPs加载质粒DNA可以在细胞中进行表达。综上表明，本研究成功制备了MCS以及具有递送核酸疫苗能力的MCS-PLGA-NPs，为FMDV核酸疫苗的递送研究提供了新的方向和见解，也为该递送载体携带特定抗原靶向抗原递呈细胞表面甘露糖受体以及应用于动物免疫的研究奠定基础。     

程序性细胞死亡因子10抑制Ⅰ型干扰素表达并促进FMDV复制

旨在探究宿主蛋白程序性细胞死亡因子10（programmed cell death factor 10，PDCD10）通过抑制Ⅰ型干扰素表达进而促进口蹄疫病毒（foot-and-mouth disease virus，FMDV）的复制。首先，本研究验证了过表达和沉默PDCD10对FMDV复制的影响，接着利用双荧光素酶报告系统探究PDCD10对Ⅰ型干扰素信号通路活化的影响，最后，利用实时荧光定量PCR探究PDCD10对Ⅰ型干扰素通路下游刺激基因（IFN-stimulated genes，ISGs）转录的影响。结果表明，过表达PDCD10显著促进FMDV的复制，沉默PDCD10显著抑制FMDV的复制。与对照相比，过表达PDCD10后感染仙台病毒（Sendai virus，SeV）的细胞培养液上清液显著促进FMDV复制，进一步，PDCD10显著抑制SeV诱导的IFN-β启动子以及NF-κB的激活且呈剂量依赖性，并且PDCD10负调控Ⅰ型干扰素通路信号分子转录，最后还发现PDCD10负调控Ⅰ型干扰素下游ISGs转录。本研究结果为深入探究PDCD10在抗病毒天然免疫中的作用积累了资料。     

Advance on Foot-and-mouth Disease Virus

Foot-and-mouth disease (FMD) is an acute,febrile and highly contagious animal disease caused by foot-and-mouth disease virus (FMDV),and has been recognized as the most important constraint to international trade in animals and animal products.An outstanding feature for FMDV infection is that the FMDV infected animals may remain as a carrier state,some of the animals exposed to FMDV may have a long term asymptomatic infection.This article will review the advance of FMDV in the following aspects,epidemiology,etiology and pathogenesis.     

Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10^-3 to 3×10^-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.     

口蹄疫病毒抗原决定簇融合基因转化苜蓿

摘要：通过农杆菌的介导,以携带O型和A型口蹄疫抗原决定簇融合基因O21 O14 A21 HBcAg的植物表达载体转化苜蓿(Medicago sativa)愈伤组织,并再生得到抗性植株。试验建立了苜蓿甘农1号愈伤组织再生系统;优化了农杆菌介导的苜蓿遗传转化系统;获得抗性再生植株4棵;GUS报告基因在转化的愈伤组织中得到瞬时表达;抗性植株PCR检测结果证实目的基因已经成功整合到苜蓿基因组中。     

南非II型口蹄疫间接ELISA检测方法的建立

为满足口岸对南非II型口蹄疫检测、监测的需求,在对南非II型口蹄疫抗原表位分析及相关基因合成的基础上,进行融合蛋白pGEX—VP1-VP3的体外表达,采用柱上酶切方法制备VP1-VP3蛋白。将纯化VP1-VP3抗原包被96微孔板,分别优化抗原包被浓度、血清稀释度、酶标二抗稀释度,确定阴性临界值。经反复优化,建立的南非II型间接ELISA检测方法的最佳抗原包被浓度为6．4ug／ml,血清最佳稀释度为1：200,酶标二抗最佳稀释度为1：4000,阴性临界值（0D450）为0．562。检测样品判定标准为：OD450〉0．622为阳性,OD450〈0．502为阴性,0．502≤OD450≤0．622为可疑。特异性试验结果表明,所建立方法与其它血清型口蹄疫间无交叉反应。南非II型口蹄疫间接ELISA检测方法的建立为我国口岸口蹄疫检疫提供了新的查验方法。     

Insertion site of FLAG on foot-and-mouth disease virus VP1 G-H loop affects immunogenicity of FLAG

The G-H loop of the foot-and-mouth disease virus(FMDV) virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid(RGD) motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker(DYKDDDDK) was inserted upstream(–4) or downstream(+10) of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites(RGD–4 and RGD+10) tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals(DIVA).     

基于免疫层析试纸的猪O型FMDV抗体水平监测

为了应用免疫层析试纸评价猪口蹄疫O型合成肽疫苗的免疫效果,对50头试验仔猪进行免疫注射,并在首次免疫后28 d及2次免疫后20 d采血,通过试纸定量测定结果来监测试验猪抗体水平变化,同时对比猪口蹄疫病毒VP1结构蛋白抗体酶联免疫吸附试验诊断试剂盒的检测结果.结果表明,猪口蹄疫O型合成肽疫苗能快速诱导产生高水平的抗体,首次免疫抗体阳性率达88%,2次免疫抗体阳性率高达100%.试纸定量测定结果表明,2次免疫后猪群免疫抗体水平整体显著提升,并保持在较高水平.与ELISA试剂盒比较二者具有相同的敏感性和特异性,符合率为100%.     

Evaluation of infectivity and transmission of different Asian foot-and-mouth disease viruses in swine

Most isolates of foot-and-mouth disease virus (FMDV) display a broad host range. Since the late 1990s, the genetic lineage of PanAsia topotype FMDV serotype O has caused epidemics in the Far East, Africa, the United Kingdom, France, the Netherlands, and numerous other countries throughout Europe and Asia. In contrast, there are several FMDV isolates that exhibit a more restricted host range. A Cathay topotype isolate of FMDV serotype O from the 1997 epizootic in Taiwan (O/TAW/97) demonstrated restricted host specificity, only infecting swine. Methods used to evaluate infectivity and pathogenicity of FMDV isolates in cattle are well-documented, but there has been less progress studying transmission and pathogenicity of FMDV isolates in pigs. In previous studies designed to examine pathogenicity, various chimeric viruses derived from O/TAW/97 were intradermally inoculated in the heel bulb of pigs. Subsequent quantitative scoring of disease and evaluation of virus released into nasal secretions and blood was assessed. Here we prove the usefulness of this method in direct and contact inoculated pigs to evaluate infectivity, pathogenicity and transmission of different Asian FMDV isolates. Virus strains within the Cathay topotype were highly virulent in swine producing a synchronous disease in inoculated animals and were efficiently spread to in-contact naive pigs, while virus strains from the PanAsia topotype displayed more heterogeneous properties.     

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%.