口蹄疫病毒的分子生物学和新型疫苗的研制

口蹄疫目前在世界上特别是在亚、非、拉美地区仍然是构成严重经济问题的一个病害,许多国家在大力研制和开发高效、安全的疫苗,包括用遗传工程手段制备的疫苗。近年来由于运用快速的核酸序列分析、重组DNA和单克隆抗体等新技术,使我们对口蹄疫病毒的基因组结构和抗原结构有了更多的了解,这对研制新型疫苗有直接的指导意义;另一方面,合成多肽的应用在模拟病毒抗原决定子及开发新型疫苗上也正崭露头角。     

猪O型口蹄疫病毒细菌样颗粒疫苗的制备与免疫原性鉴定

验证基于革兰氏阳性增强基质(Gram-positive enhancer matrix,GEM)展示口蹄疫病毒细菌样颗粒(Bacteria-like particles,BLP)疫苗的可行性。按照大肠杆菌偏好性密码子优化合成基于猪口蹄疫病毒Mya98株序列的3种抗原基因设计,并将其插入到含有锚钩蛋白基因的原核表达载体p QZ-PA,鉴定阳性后转入Escherichia coli BL21,进行诱导表达。利用SDS-PAGE与Western blotting对目的基因表达及产物的可溶性进行分析。利用GEM颗粒纯化目的蛋白,制备细菌样颗粒疫苗抗原;利用BCA试剂盒测定重组蛋白的浓度,将重组蛋白与白油佐剂乳化,制备疫苗,免疫5周龄小鼠,同时设商品化多肽苗对照与空白对照,免疫后不同时间采集试验小鼠血清,利用口蹄疫病毒多肽ELISA抗体检测试剂盒和O型口蹄疫抗体液相阻断酶联免疫(Enzyme-linked immunosorbent assay,ELISA)检测试剂盒检测免疫小鼠血清的抗体水平;利用噻唑蓝比色法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)测定淋巴细胞增殖情况;利用荧光定量PCR方法检测相关细胞因子表达,评价细胞免疫水平。SDS-PAGE结果表明,设计在大肠杆菌中的3种口蹄疫病毒抗原基因均以可溶形式获得高效表达;Western blotting结果显示,表达的重组蛋白能够与口蹄疫病毒阳性血清发生反应,利用GEM颗粒能够实现重组蛋白的一步离心纯化,制备BLP疫苗抗原;免疫试验结果表明,设计的重组抗原B(T1BT2)4B不但能够刺激免疫小鼠产生更高水平的多肽特异性ELISA抗体与口蹄疫特异性液相阻断抗体,而且产生了更高水平的脾淋巴细胞增殖及Th1型的细胞因子分泌。初步实验结果表明,本研究制备的BLP疫苗GEM-B(T1BT2)4B具有良好的免疫原性,为研究口蹄疫病毒基因工程亚单位疫苗开辟了一条新的思路。     

植物中表达口蹄疫病毒抗原研究进展

口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)引起的一种急性、烈性、高度接触性传染病,严重危害畜牧养殖业健康发展。口蹄疫灭活疫苗是口蹄疫防控的主导产品,为控制口蹄疫流行起到了重要作用;但是也存在抗原不稳定、在生产制备过程中存在因病毒灭活不彻底而散毒的风险、生产成本较高等问题。与传统的微生物和动物生物反应器相比,通过转基因技术以植物作为生物反应器生产抗原蛋白,具有成本低廉、安全便捷、易于储运等一些优势,且无需蛋白提取纯化过程,可直接食用免疫;但也存在着表达量低、控制性差等问题。因此,通过植物生物反应器表达口蹄疫病毒抗原蛋白,可能是一种具有一定优势但仍需不断优化的疫苗生产手段。本文综述了在植物中表达活性蛋白的主要策略,以及通过植物生物反应器表达口蹄疫病毒抗原蛋白的研究进展,并讨论了目前面临的问题与挑战,以期为相关工作提供一定的借鉴和参考。     

口蹄疫反向疫苗研究进展

利用反向遗传技术对口蹄疫病毒基因的改造和修饰,可以实现对疫苗种毒的生产性能、抗原匹配性、抗原稳定性、免疫应答能力、生物安全性等特征的改良,能够迅速获得预期生物学特性的疫苗候选株,进而可以减少和避免流行毒株驯化环节带来的负面影响。不仅改变了对流行毒株筛选驯化受病毒自然属性制约、费时费力、成功率低的缺陷,而且可以实现更为主动有效的疫苗毒株的设计,对整体提升疫苗品质和效力具有重大意义。本文以口蹄疫病毒为例,从改良疫苗毒株的生物学特性方面进行了综述,为相关研究提供参考和借鉴。     

用口蹄疫病毒非结构蛋白区分注苗动物和自然感染动物研究进展

口蹄疫是由口蹄疫病毒引起的偶蹄类动物烈性传染病,疫苗接种是防治口蹄疫暴发的主要措施之一,而要控制口蹄疫流行,首先要将病毒感染动物从疫苗接种群体中区分开来。以口蹄疫病毒非结构蛋白（NSP）为抗原,检测动物体内的NSP抗体是一种很好的区分感染动物和疫苗免疫动物的诊断方法,许多实验室开展了相关研究,并取得了一定的成绩。     

保护剂及其在口蹄疫疫苗中的研究进展

保护剂在动物疫苗中具有重要的作用,直接影响疫苗的质量.以保护剂为核心介绍了在疫苗保存前病毒抗原和保护剂的准备,保护剂的选用原则,分类、作用机理,着重讨论了保护剂在口蹄疫疫苗中的研究进展以及当前存在的问题,并对其应用前景提出展望.     

Asia I口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia I口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法。用纯化的Asia I型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠, 将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合, 采用间接ELISA和有限稀释法筛选杂交瘤细胞。分别用ELISA、Western blotting检测mAb腹水的效价及其特异性。筛选到杂交瘤细胞2株, 腹水效价均在100×29以上; 以纯化后的Asia I型口蹄疫病毒vp2重组蛋白作为抗原, 利用Asia I型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia I型口蹄疫抗体。临床应用表明, 该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%, 和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%。     

转基因植物作为生物反应器生产口蹄疫疫苗的研究进展

利用转基因植物作为生物反应器表达重组蛋白,生产外源蛋白质作为动物疫苗是一个很有吸引力的廉价生产系统,它有可能代替生产成本较高的传统疫苗的发酵生产系统。通过口蹄疫病毒VPI结构蛋白基因在转基因植物中的表达,口蹄疫疫苗已在植物中产生。在植物中生产的抗原能够保持其自身的免疫原性。本文简要综述了近十年来用转基因植物作为生物反应器生产口蹄疫疫苗的研究进展、特点及其应用前景。     

Asia Ⅰ口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia Ⅰ口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法.用纯化的Asia Ⅰ型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠,将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合,采用间接ELISA和有限稀释法筛选杂交瘤细胞.分别用ELISA、Western blotting检测mAb腹水的效价及其特异性.筛选到杂交瘤细胞2株,腹水效价均在100×2<'9>以上:以纯化后的AsiaⅠ型口蹄疫病毒vp2重组蛋白作为抗原,利用Asia Ⅰ型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia Ⅰ型口蹄疫抗体.临床应用表明,该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%,和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%.     

口蹄疫病毒非结构蛋白3AB双抗体夹心ELISA方法的建立

抗原纯净度是口蹄疫(Foot-and-mouthdisease,FMD)灭活疫苗质量检验的一项重要内容,一般采用疫苗2–3次免疫动物后,检测非结构蛋白(Non-structuralprotein,NSP)抗体是否阳转,判断疫苗抗原的纯净度。文中旨在建立定量检测FMD灭活疫苗抗原中NSP3AB含量的ELISA方法,为疫苗质量控制提供参考方法。利用口蹄疫病毒(Foot-and-mouthdiseasevirus,FMDV)NSP3A单克隆抗体和辣根过氧化物酶(Horseradish peroxidase,HRP)标记的3B单克隆抗体,建立定量检测NSP3AB含量的双抗体夹心ELISA检测方法。采用原核表达并纯化的3AB蛋白作为标准品,标准品系列稀释,绘制标准曲线,以标准品与未加抗原的阴性对照吸光值(OD)的比值大于2.0的标准品最低浓度为最低检测限。标准品浓度介于4.7–600.0 ng/mL之间时,测得的OD值与浓度呈线性相关,回归曲线呈直线,相关系数R2=0.99,确定最低检测限为4.7ng/mL。检测12份未纯化灭活抗原中3AB蛋白含量介于9.3–200.0ng/mL之间;而纯化后的...     

A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites; no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.     

口蹄疫病毒结构蛋白P1-2A及蛋白酶3C基因的转基因番茄对豚鼠免疫反应的初步研究

构建了O型口蹄疫病毒China99株结构蛋白P1-2A、非结构蛋白3C以及部分2B基因(P1-2X3C)的植物双元表达载体pBin438/P1-2X3C,通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得40余株抗性植株,对得到的抗性植株进行分子生物学检测,65%的再生植株PCR检测阳性;RT-PCR结果证实P1-2X3C基因在转基因番茄中能够有效转录;ELISA和Western blot检测表明转基因植株中表达的目的蛋白具有免疫反应性。转基因番茄叶片蛋白粗提液经肌肉途径免疫豚鼠,于第3次免疫后28d用100ID50/0.2mL的同源强毒攻击,结果表明口蹄疫病毒P1-2X3C基因的转基因番茄表达产物具有良好的免疫原性,豚鼠3免后血清效价可达1:64~1:128,攻毒后两组免疫豚鼠保护率分别达3/5和5/5。     

Single amino acid substitution of VP1 N17D or VP2 H145Y confers acid-resistant phenotype of type Asia1 foot-and-mouth disease virus

Infection by foot-and-mouth disease virus (FMDV) is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. However, dissociation of the FMDV 146S particle in mildly acidic conditions renders inactivated foot-and-mouth disease (FMD) vaccines much less effective. Type Asia1 FMDV mutants with increased resistance to acid inactivation were selected to study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of FMDV. Sequencing of capsid-coding regions revealed four amino acid replacements (VP1 N17D, VP2 H145Y, VP2 G192D, and VP3 K153E) in the viral population of the acid-selected 10th passage. We performed single or combined mutagenesis using a reverse genetic system, and our results provide direct experimental evidence that VP2 H145Y or VP1 N17D substitution confers an acid-resistant phenotype to type Asia1 FMDV.     

Monitoring sequence space as a test for the target of selection in viruses

An essential feature of viral quasispecies, predicted from quasispecies theory, is that the target of selection is the mutant distribution as a whole. To test molecularly the mutant composition selected from a viral quasispecies we reconstructed a mutant distribution using 19 antigenic variants of foot-and-mouth disease virus (FMDV). Each variant was marked by a specific amino acid replacement at a major antigenic site of the virus that conferred resistance to a monoclonal antibody (mAb). The variants were introduced in the mutant spectrum of a biological FMDV clone, at a frequency commonly found in FMDV quasispecies. The reconstructed quasispecies (and a number of control populations) were allowed to replicate in the presence or absence of the mAb. The mutant distribution that became dominant as a result of antibody selection included at least ten of the 19 mutants initially used to reconstruct the quasispecies. No such biased mutant repertoire was found in control populations. The results show that a mutant distribution was selected, and are incompatible with selection of an individual genome, which then generated multiple mutants upon further replication. An ample representation of variants immediately following a selection event should contribute to subsequent adaptability of the virus.     

2017–2022年中国口蹄疫流行状况分析

【目的】分析近年来中国口蹄疫流行和传播特点,研判口蹄疫流行趋势。【方法】以2017-2022年间中国报告发生的口蹄疫疫情为研究对象,从疫情的“三间分布”、生产环节分布、流行毒株分子流行病学分析及溯源等方面,对近6年的疫情情况进行系统梳理。【结果】2017-2022年间中国共有15个省份报告发生口蹄疫疫情54次。总体形势稳定：Asia 1型口蹄疫维持无疫状态;2019-2022年连续4年未发生A型口蹄疫疫情;田间以散发O型口蹄疫为主。六年间报告牛口蹄疫疫情36次,羊疫情1次,猪疫情17次。分子流行病学研究表明,O/Mya-98、O/Ind-2001、O/CATHAY、O/PanAsia和A/Sea-97这5个流行毒株同时流行,且与同时期周边国家（缅甸、老挝和越南等）口蹄疫毒株遗传关系密切。流行病学调查结果显示,疫情（尤其是牛疫情,占比66.7%）主要发生在流通（57%）、散养（32%）等免疫薄弱环节。【结论】对内强化疫苗免疫和流通动物管控,对外严防境外毒株传入仍是今后中国口蹄疫防控的重要任务。     

Development of high-affinity single chain Fv against foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (K_D = 42.7 nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV.     

口蹄疫O型重组病毒的构建及其免疫原性分析

【目的】利用反向遗传操作技术,构建含O型口蹄疫病毒(food-and-mouth disease virus, FMDV) 3个拓扑型免疫优势结构蛋白基因的重组FMDV,评估其作为猪O型口蹄疫(food-and-mouth disease, FMD)疫苗候选株的潜力。【方法】通过基因合成,在FMD疫苗株O/HN/CHA/93 (古典中国拓扑型)的基因中嵌合流行株O/NXYCh/CHA/2018 (东南亚拓扑型) VP1结构蛋白的重组病毒骨架上,用O/TUR/5/2009疫苗株(中东-南亚拓扑型) VP1蛋白的G-H环基因替换其对等基因,构建含O型3个拓扑型FMDV结构蛋白基因的重组全长质粒,Not I线性化后转染表达T7 RNA聚合酶的BSR/T7细胞,拯救重组病毒。通过RT-PCR、序列测定、间接免疫荧光鉴定重组病毒;噬斑试验和一步生长曲线分析重组病毒的生物学特性。重组病毒制备疫苗免疫猪,用病毒中和试验分析其对当前流行的O型3个拓扑型FMDV的交叉反应性。【结果】成功拯救到含O型3个拓扑型FMDV结构蛋白基因的重组病毒,重组病毒与亲本病毒具有相似的生物学特性。亲本病毒和重组病毒制备的疫苗免疫猪,均能够对中东-南亚型(Middle East-South Asia, ME-SA)拓扑型和东南亚型(South-East Asia, SEA)拓扑型病毒株产生保护性平均中和抗体(>1.65log₁₀);均不能对古典中国型(Cathay)拓扑型流行株产生保护性平均中和抗体(<1.65log₁₀),但与亲本病毒相比,O/TUR/5/2009疫苗株G-H环基因的替换显著提高了对ME-SA和SEA拓扑型病毒株的交叉反应性(p<0.05)。【结论】本研究对未来FMD疫苗的设计具有重要的指导意义。