| 美洲商陆抗病毒蛋白真核表达载体的构建及在毕赤酵母中的表达 |
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| 引用本文： | 陈定虎,王锡锋,李莉,周广和.美洲商陆抗病毒蛋白真核表达载体的构建及在毕赤酵母中的表达[J].农业生物技术学报,2003,11(2):183-186. |
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| 作者姓名： | 陈定虎 王锡锋 李莉 周广和 |
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| 作者单位： | 中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京,100094 |
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| 基金项目： | 农业部"948"项目(991003). |
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| 摘 要： | 摘要：从美洲商陆（Phytolacca americana）叶片中通过异硫氰酸胍法提取出了总RNA,经RT-PCR扩增出缺失突变型抗病毒蛋白PAP基因,将该基因克隆于分泌型真核表达载体pPIC9K,然后导入毕赤酵母(Pachia pastoris )菌株GS115细胞。在甲醇的诱导下,经过酵母高密度发酵进行PAP的表达,经SDS-PAGE分析。结果表明,在培养基上清液中含有一明显的特异性蛋白条带,大小为34 kD,经Western-blotting分析,该蛋白与法国PAP抗血清有特异性反应,体外活性检测表明该蛋白对病毒的侵染性具有高度的抑制性,表明该基因在毕赤酵母GS115中得到了表达。
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| 关 键 词： | 关键词：美州商陆抗病毒蛋白 毕赤酵母 发酵 表达 活性测定 |
| 修稿时间： | 2002年5月15日 |
A Secreted Expression Vector Construction of Pokeweed Antiviral Protein Gene and Its Expression in Pachia pastoris |
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| Abstract: | Abstract：The total RNA was isolated from pokeweed (Phytolacca americana ) leaves using the method of guanidine isothiocyante and used as template for amplification of the deleted mutant pokeweed antiviral protein (PAP ) gene by RT-PCR and then the amplified fragment was cloned into secreted expression pPIC9K vector to form pPIC9K-p, then the vector was transferred into Pachia pastoris GS115 strain. The specific expression protein was secreted into the medium after inducing with methanol and the protein amount reached about 50~60μg /mL measured by UV-absorbed methods in the supernatant of the medium via high density fermentation. SDS-PAGE results showed that there was one main protein band with molecular weight about 34 kD in the fermentation supernatant and it could specifically react with France PAP antiserum in Western blotting. Activity tests revealed that this protein could inhibit plant virus infection with high efficiency. |
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