Asia1型口蹄疫新型多表位疫苗免疫豚鼠的效力试验

摘要

目的：评价一种改进型A sia 1型口蹄疫多表位重组疫苗的免疫效能。方法根据A sia 1型口蹄疫病毒的基因序列设计多表位基因，构建重组表达质粒（pRE-oIgG）。用大肠杆菌细胞分别表达靶标蛋白RE-oIgG和口蹄疫病毒3D蛋白，并用组氨酸亲和层析柱纯化。纯化的RE-oIgG ，3D和RE-oIgG与3D的混合物分别经油性佐剂IAS206乳化后配制成相应的疫苗。25只雌性豚鼠被随机分成5组，每组5只豚鼠，经肌肉途径分别注射RE-oIgG、3D、RE-oIgG与3D的混合物、Asia 1口蹄疫灭活疫苗和PBS ，所有动物进行两次免疫。采用ELISA、病毒微量中和试验、攻毒试验和流式细胞术分别测定免疫前后抗口蹄疫特异性抗体、中和抗体、保护率以及淋巴细胞增殖反应。结果 RE-oIgG与3D的混合物能够诱导高效价的抗Asia 1型口蹄疫病毒的特异性抗体，与RE-oIgG单独免疫豚鼠相比较具有明显的差异性（P＜0．05）。除了阴性对照组PBS外，来自所有免疫动物的外周血淋巴细胞经过体外培养刺激后均产生了明显的淋巴细胞增殖反应。单独免疫3D蛋白和安慰剂PBS实验组既没有测到抗口蹄疫病毒特异性抗体，也没有测到中和抗体。而RE-oIgG与3D的混合物不仅诱导机体产生了高水平的保护性中和抗体，而且诱发了明显的淋巴细胞增殖反应。更为重要的是，该蛋白质混合物针对1000 GPID50强毒攻击后，所有动物没有出现症状完全保护。值得一提的是，单独免疫3D蛋白的5只豚鼠中，其中2只完全保护；另外3只虽然出现了临床症状，但相对PBS对照组来说发病时间大约推迟2～3 d。因此，笔者认为RE-oIgG与3D的混合物不仅能够诱导体液免疫反应，而且能够诱发细胞免疫反应，是一种高效能的新型疫苗，将来可用于我国口蹄疫的防控。%The potency of an improved recombinant multi-epitope vaccine against FMDV type Asia1 was evaluated in this study .A multi-epitope gene based on FMDV type Asia1 was designed and a recombinant expression plasmid (pRE-oIgG) was constructed .The proteins ,RE-oIgG and 3D were expressed in E .coli cells and purified with Ni-NTA agarose resin by affinity chromatography .The proteins ,RE-oIgG ,3D and RE-oIgG plus 3D ,were emulsified in an oil adjuvant ISA 206 .Twenty-five female guinea pigs were randomly divided into five groups and intramuscularly vaccinated for with RE-oIgG ,3D ,RE-oIgG plus 3D ,an inactivated FMDV vaccine (type Asia1) ,and PBS .All animals were vaccinated for two times .Anti-FMDV specific an-tibodies ,neutralization antibodies ,protection potency ,and lymphoproliferation assay were detected by ELISA ,virus neutrali-zation assay ,challenge test ,and flow cytometry ,respectively .Results showed that RE-oIgG plus 3D elicited significant high-level anti-FMDV specific antibodies compared to RE-oIgG alone (P<0 .05) .All the vaccinated animals induced higher level lymphoproliferation responses in vitro except PBS .Both 3D alone and PBS produced the negligible neutralizing antibodies and anti-FMDV specific antibodies .RE-oIgG plus FMDV 3D not only elicited high levels of anti-FMDV neutralizing antibodies ,but also induced significant lymphoproliferation responses .More importantly ,RE-oIgG plus 3D conferred complete protection to guinea pigs against challenge with 1 000 GPID50 .Interestingly ,two of five vaccinated animals with 3D alone were full protected against challenge ,and other three animals significantly showed a delay of 2-3 days in the onset of clinical signs .Therefore ,we considered that RE-oIgG plus 3D induces strong humoral and cellular immune responses ,which may be used for control and prevention of FMD in the future .