摘要
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。
The SRSV antigenic protein was expressed in the broad bean plants by Potyvirus vector pCIYVV/CP/W. The fulllength sequence of 1605 bp for SRSV was inserted between nuclear inclusion b （NIb） and coat protein （CP） of infectious viral vector pCIYVV/CP/W derived from clover yellow vein virus （CIYVV） of potyvirus. The recombinant plasmids were constructed and named pCIYVV-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 CIYVV. The genetic stability of the reconlbinant 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 coll. The results showed that these proteins post expressing and automatically separated from the CIYVV pro-proteins were stable even in following subsequential passages of the progeny rccombinants in broad bean plants.
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2006年第1期35-39,共5页
Journal of Shenyang Agricultural University
基金
辽宁省教育厅资助项目(05L933)
