FMDV中和表位在HBc上的展示及其嵌合蛋白在重组腺病毒中的表达
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摘要
口蹄疫(Foot-and-mouth disease, FMD)是主要感染偶蹄动物的一种急性、高度接触性传染病。该病以其流行广、传播快、危害大的特点成为威胁偶蹄动物的首位传染病。鉴于口蹄疫所带来的严重的经济和社会影响,国际兽医局(OIE)将其列为必须通报的疾病之一,我国也将其列为一类动物传染病的第一位。口蹄疫的病原为口蹄疫病毒(Foot-and-mouth disease Virus, FMDV),是小核糖核酸病毒科口蹄疫病毒属的唯一成员,共包括O、A、C、SAT1、SAT2、SAT3、Asia1型七个血清型,不同血清型间无交叉保护。该病毒的高度变异性是口蹄疫难于防控的主要原因。
在我国,疫苗接种是防控FMD的主要手段。虽然目前广泛应用的全病毒灭活苗具有免疫原性强,保护力好等优点,但在应用过程中,也发现其具有免疫期短、副反应强及存在安全隐患等诸多缺陷,这使FMDV新型疫苗成为当今的研究热点,其中,利用病毒样颗粒展示FMDV抗原表位的表位展示疫苗,因其具有天然的抗原递呈特征、安全性好等优势而展现出广阔的开发前景。
本研究选择乙肝核心抗原(Hepatitis B Virus core antigen, HBc)蛋白骨架作为载体,将Asia1型和O型FMDV的中和表位设计成特定的展示结构插入其中,由此构建的嵌合蛋白以pcDNA3.1(+)载体在真核细胞中瞬时表达,利用HBc的自组装特性,使特定结构的表位展示于病毒样颗粒的表面,通过间接免疫荧光试验检测展示表位的抗原活性。结果显示,将Asia1型FMDV模拟表位展示于HBc的N末端、将O型FMDV的真实表位单独展示在HBc的MIR区和N末端时,与表位相应的单克隆抗体呈现强反应,说明设计的表位结构已经得到正确的展示。随后,本研究又构建了在HBc的MIR区和N端同时插入FMDV表位的双表位展示载体以及表达该嵌合蛋白的重组腺病毒,结果表明,利用重组腺病毒表达双表位的表位-HBc嵌合蛋白不但实现了理想的表达效果、嵌合蛋白与相应单抗也呈现较强的反应活性。
本研究通过在HBc上展示FMDV的中和表位,以及将表位与HBc嵌合蛋白在腺病毒载体中表达,为FMDV表位展示疫苗的研制建立了理论与实验基础。
Foot-and-mouth disease (FMD) is an acute and highly contagious disease affecting cloven-hoofed animals and is characterized by widespread epidemicity, rapid transmission and great harm. Severe economic and social impacts make FMD to be a first disease listed by the World Organisation for Animal Health (OIE). In our country, it is also listed as the first one of Class I animal infectious diseases. The foot-and-mouth disease virus (FMDV) is the prototype member of the genus Aphthovirus within the family Picornaviridae. Seven distinct serotypes of the virus have been defined (O, A, C, Asial and SAT-1,2 and 3) and there is no cross-protection among serotypes.The variability of this genome is the main reason why it is difficult to prevent and control FMD.
In China, vaccination is the main means of preventing and controlling FMD. Nowadays, genetically engineered vaccine become a research focus, because inactivated whole-virus vaccines that are widely used have some defects such as short duration of immunity, serious side effects and potential safety hazard. Alternative approaches have been investigated to control the disease in which vaccines based on epitopes displayed on the Virus-like Particles were expected to overcome present defects and showed bright prospects.
In this study, Hepatitis B Virus core antigen (HBc) was choosed as skeleton carrier to display the neutralizing epitopes of FMDV by specific structures. Recombinant HBc spontaneously assembled into Virus-like Particles and the epitopes displayed on the surface of particles through expressing the chimeric protein in eukaryocyte. Then the reactivity of displayed epitope was detected by immunofluorescence assay (IFA). The experimental results indicated that the the best immunoreactive mimotope of Asial-type FMDV inserted in the N-terminal of HBc had high reactivity with McAb, and the neutralizing epitope of O-type FMDV inserted on MIR or N-terminal of HBc also had high reactivity with McAb. These results showed that the epitopes were display on the surface of HBc with correct conformation. Then the chimeric proteins of double epitopes and HBc were expressed well by recombinant advenvirus and had high reactivity with McAb.
All the research above laid the theoretical and experimental foundation for research of epitope-displaying vaccine.
在我国,疫苗接种是防控FMD的主要手段。虽然目前广泛应用的全病毒灭活苗具有免疫原性强,保护力好等优点,但在应用过程中,也发现其具有免疫期短、副反应强及存在安全隐患等诸多缺陷,这使FMDV新型疫苗成为当今的研究热点,其中,利用病毒样颗粒展示FMDV抗原表位的表位展示疫苗,因其具有天然的抗原递呈特征、安全性好等优势而展现出广阔的开发前景。
本研究选择乙肝核心抗原(Hepatitis B Virus core antigen, HBc)蛋白骨架作为载体,将Asia1型和O型FMDV的中和表位设计成特定的展示结构插入其中,由此构建的嵌合蛋白以pcDNA3.1(+)载体在真核细胞中瞬时表达,利用HBc的自组装特性,使特定结构的表位展示于病毒样颗粒的表面,通过间接免疫荧光试验检测展示表位的抗原活性。结果显示,将Asia1型FMDV模拟表位展示于HBc的N末端、将O型FMDV的真实表位单独展示在HBc的MIR区和N末端时,与表位相应的单克隆抗体呈现强反应,说明设计的表位结构已经得到正确的展示。随后,本研究又构建了在HBc的MIR区和N端同时插入FMDV表位的双表位展示载体以及表达该嵌合蛋白的重组腺病毒,结果表明,利用重组腺病毒表达双表位的表位-HBc嵌合蛋白不但实现了理想的表达效果、嵌合蛋白与相应单抗也呈现较强的反应活性。
本研究通过在HBc上展示FMDV的中和表位,以及将表位与HBc嵌合蛋白在腺病毒载体中表达,为FMDV表位展示疫苗的研制建立了理论与实验基础。
Foot-and-mouth disease (FMD) is an acute and highly contagious disease affecting cloven-hoofed animals and is characterized by widespread epidemicity, rapid transmission and great harm. Severe economic and social impacts make FMD to be a first disease listed by the World Organisation for Animal Health (OIE). In our country, it is also listed as the first one of Class I animal infectious diseases. The foot-and-mouth disease virus (FMDV) is the prototype member of the genus Aphthovirus within the family Picornaviridae. Seven distinct serotypes of the virus have been defined (O, A, C, Asial and SAT-1,2 and 3) and there is no cross-protection among serotypes.The variability of this genome is the main reason why it is difficult to prevent and control FMD.
In China, vaccination is the main means of preventing and controlling FMD. Nowadays, genetically engineered vaccine become a research focus, because inactivated whole-virus vaccines that are widely used have some defects such as short duration of immunity, serious side effects and potential safety hazard. Alternative approaches have been investigated to control the disease in which vaccines based on epitopes displayed on the Virus-like Particles were expected to overcome present defects and showed bright prospects.
In this study, Hepatitis B Virus core antigen (HBc) was choosed as skeleton carrier to display the neutralizing epitopes of FMDV by specific structures. Recombinant HBc spontaneously assembled into Virus-like Particles and the epitopes displayed on the surface of particles through expressing the chimeric protein in eukaryocyte. Then the reactivity of displayed epitope was detected by immunofluorescence assay (IFA). The experimental results indicated that the the best immunoreactive mimotope of Asial-type FMDV inserted in the N-terminal of HBc had high reactivity with McAb, and the neutralizing epitope of O-type FMDV inserted on MIR or N-terminal of HBc also had high reactivity with McAb. These results showed that the epitopes were display on the surface of HBc with correct conformation. Then the chimeric proteins of double epitopes and HBc were expressed well by recombinant advenvirus and had high reactivity with McAb.
All the research above laid the theoretical and experimental foundation for research of epitope-displaying vaccine.
引文
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