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目的研发一种具有良好免疫原性的H3N8型马流感血凝素(hemagglutinin,HA)核酸疫苗。方法根据马A型流感病毒A/Equine/Xinjiang/3/08(H3N8)的HA蛋白序列,经密码子优化后化学合成能够表达相应蛋白的基因片段,并将该片段克隆到核酸疫苗载体pJW4303中,构建带有天然信号肽的H3HA核酸疫苗,命名为H3HA/XJ3-08-wt;进一步对HA基因进行修饰,以人组织纤维蛋白酶原激活剂(human tissue plasminogen activator,tPA)取代H3HA天然信号肽,命名为H3HA/XJ3-08-tPA,或切除部分HA的基因片段使之只表达HA蛋白的胞外域,命名为H3HA/XJ3-08-dTM。用这3种重组质粒分别转染293T细胞,蛋白表达经蛋白质印迹检测得到确认。采用电转录法免疫新西兰白兔。ELISA方法检测免疫后兔血清中抗H3HA抗体滴度,血凝抑制实验(hemagglutination inhibition,HI)检测保护性抗体水平。结果 3种H3HA核酸疫苗均可在293T细胞中高效表达,并能够在新西兰白兔体内诱导产生特异性抗H3HA抗体,HI检测到不同水平的保护性抗体。其中以H3HA/XJ3-08-tPA的免疫原性最强。结论新构建的H3N8型马流感血凝素核酸疫苗具有良好的免疫原性,为此类疫苗的进一步研发奠定了基础。
Objective To develop a highly immunogenic H3N8 influenza virus hemagglutinin (HA) DNA vaccine. Methods Based on the HA protein sequence of A / Equine / Xinjiang / 3/08 (H3N8), the gene fragment encoding the corresponding protein was chemically synthesized after codon optimization. The fragment was cloned into DNA vaccine vector pJW4303 , A H3HA DNA vaccine with a native signal peptide was constructed and named as H3HA / XJ3-08-wt. The HA gene was further modified to replace the H3HA native signal peptide by human tissue plasminogen activator (tPA) , Named H3HA / XJ3-08-tPA, or excised a portion of the HA gene fragment to express only the HA protein extracellular domain, designated as H3HA / XJ3-08-dTM. 293T cells were transfected with the three recombinant plasmids, and the protein expression was confirmed by Western blot. New Zealand white rabbits were immunized by electrotransformation. Anti-H3HA antibody titers were detected by ELISA in the serum of rabbits after immunization. Protective antibody was detected by hemagglutination inhibition (HI). Results All three H3HA DNA vaccines were highly expressed in 293T cells and were able to induce specific anti-H3HA antibodies in New Zealand white rabbits. Different levels of protective antibodies were detected in HI. Which H3HA / XJ3-08-tPA the strongest immunogenicity. Conclusion The newly constructed H3N8 influenza virus hemagglutinin nucleic acid vaccine has good immunogenicity and lays the foundation for further research and development of such vaccines.