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

Immunogenicity comparison of three multi-epitope proteins expressed in Escherichia coli against swine foot-and-mouth disease virus serotype A

【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。

[Objective] A multi-epitope protein vaccine of foot-and-mouth disease virus(FMDV) serotype A was developed to provide a safe and effective candidate vaccine for the prevention and control of FMD in pigs.[Methods] We designed and synthesized multi-epitope immunogenic genes A10, IA10 and FA10 based on the results of previous experiments and the global epidemiological information of FMDV serotype A. The expressed proteins were emulsified with ISA 201 adjuvant to immunize pigs. Serum IgG antibody titers were detected by liquid phase blocking ELISA at 0, 14 and 28 days post-immunization. All pigs were challenged with virulent FMDV A/HNLY4/2014 after a single inoculation.[Results] Three recombinant proteins were successfully expressed in E. coli and specific bands of approximately 35, 57 and 64 kDa, which were consistent with the expected size of the recombinant proteins A10, IA10 and FA10, respectively. The recombinant proteins were also recognized specifically by the anti-FMDV(type A) antibodies. A10+201 induced lower level of total IgG antibodies than the inactivated vaccine, but higher than other groups. All pigs(100%) immunized with A10+201 and inactivated vaccine were completely protected against virulent FMDV challenge. Four out of five pigs(80%) immunized with IA10+201 and FA10+201 were protected and only one out of five pigs(20%) immunized with A10 and FA10 were protected. All pigs inoculated with PBS+201 were unprotected.[Conclusion] A10+201 conferred the greatest protection against FMDV serotype A challenge in pigs and can be further evaluated as a candidate vaccine.