A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

瞄准：表示 23-ku pE2 到花费有效地，肝炎 E 病毒(HEV ) 的 3'-portion 包括的 E2 碎片编码的最有希望的子单元疫苗打开读物框架 2 (ORF2 ) 在烟草的质体(Nicotiana 烟 cv。SR1 ) ，在质体调查 transgene 表示和 pE2 累积，并且评估反在老鼠的导出质体的 pE2 的遗传因子的效果。方法：包含米饭 psbA 倡导者驾驶的 E2 碎片的指向质体的向量 pRB94-E2 被构造。在进烟草质体的交货之上，这构造能在质体染色体在 psaB-trnfM 和 trnG-psbC 碎片开始相应再结合，并且导致在二碎片之间插入的 transgene。pRB94-E2 与一个 biolistic 粒子轰炸方法被交付，并且转变质体的工厂被获得在补充 spectinomycin 的媒介上跟随炮轰的叶纸巾的新生。改革工厂的 Transplastomic 地位被 PCR 和南部的污点分析证实， transgene 表示被北污点分析调查，并且 pE2 的累积被 ELISA 测量。而且，蛋白浸出物被用来使老鼠免疫，并且在使免疫的老鼠的浆液样品的 pE2 反应的抗体的存在被 ELISA 学习。结果：PCR 和南部的污点分析证实的 Transplastomic 线能活跃地抄录 E2 mRNA。pE2 多肽象 13.27 microg/g 一样高被积累到水平新鲜叶子。pE2 能刺激使免疫的老鼠产生 pE2 特定的抗体。结论：HEV-E2 碎片能被插入到质体染色体，导出的 recombinant pE2 抗原是在老鼠遗传因子的反。因此，质体可以是为 HEV 疫苗的划算的生产的新奇来源。

AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of tobacco （Nicotiana tabacum cv. SR1）, to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS： Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS： Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION： HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.