用植物病毒载体表达小球状病毒抗原蛋白的研究

将引起人类腹泻主要病原物小球状病毒(Small round structured virus;SRSV)编码抗原蛋白基因的全长序列1605bp,导入来自三叶草黄脉病毒(Clover yellow vein virus;ClYVV)侵染性全长cDNA克隆的侵染性植物病毒表达载体pClYVV/CP/W基因组的NIb/CP基因之间,构建了重组病毒克隆pClYVV-NV1.6。用上述重组病毒克隆接种蚕豆植物,表现了与野生型ClYVV相同的症状。从发病叶中提取总RNA,用反转录PCR(RT-PCR)对上述重组病毒克隆的转录进行了检测,结果表明,外源基因在F4子代病毒基因组中仍然稳定存在。从发病叶中提取总蛋白,用酶联免疫吸附分析(Enzyme-linked immunosorbent assay;ELISA)及蛋白质杂交(Western blotting;WB)对上述重组病毒克隆表达的目的基因产物抗原蛋白进行了测定,结果表明,重组病毒克隆pClYVV-NV1.6之目的基因产物的表达量随寄主植物发病后时间的推移而变化,最大表达是在寄主植物发病后第9天,最大表达量为每克鲜叶可表达160.00μg,这一研究结果为用植物生产抗SRSV口服疫苗提供了有力的基础。

Study on SRSV Antigenic Protein Expression in the Plant by Plant Virus Vector

The SRSV antigenic protein was expressed in the broad bean plants by Potyvirus vector pClYVV/CP/W.The full-length sequence of 1605 bp for SRSV was inserted between nuclear inclusion b(NIb) and coat protein(CP) of infectious viral vector pClYVV/CP/W derived from clover yellow vein virus(ClYVV) of potyvirus.The recombinant plasmids were constructed and named pClYVV-NV1.6,and then they were mechanically inoculated in broad bean seedlings.The similar apparent symptom on the plant was observed post infection(pi) of the recombinant plasmids as well as the wild-type ClYVV.The genetic stability of the recombinant plasmids carrying foreign gene was examined by RT-PCR.The expressed amounts of antigenic protein were confirmed by sandwich enzyme-linked immunosorbent assay(ELISA) and western blot analysis using monoclonal antibodies against the SRSV-G2 antigenic protein expressed in Escherichia coli.The results showed that these proteins post expressing and automatically separated from the ClYVV pro-proteins were stable even in following subsequential passages of the progeny recombinants in broad bean plants.