体外表达豇豆花叶病毒 B 株 RNA 的全长 DNA 拷贝：鉴定 B RNA 编码的 24kD 蛋白为病毒蛋白酶。
In vitro expression of a full-length DNA copy of cowpea mosaic virus B RNA: identification of the B RNA encoded 24-kd protein as a viral protease.
机构信息
Department of Molecular Biology, Agricultural University, De Dreijen 11, 6703 BC Wageningen, The Netherlands.
出版信息
EMBO J. 1987 Mar;6(3):549-54. doi: 10.1002/j.1460-2075.1987.tb04789.x.
Double-stranded cDNA was synthesized from B component RNA of cowpea mosaic virus and cloned into appropriate vectors. Using four clones, together representing the entire B RNA sequence, a full-length DNA copy was constructed and subsequently positioned downstream of a phage SP6 or T7 promoter. RNA molecules transcribed from this full-size DNA copy using SP6 or T7 RNA polymerase were efficiently translated in rabbit reticulocyte lysates into a 200-kd polypeptide similar to RNA isolated from viral B components. Moreover this polypeptide was rapidly cleaved into 32-kd and 170-kd polypeptides, exactly like the 200-kd polypeptide encoded by viral B RNA. In vitro transcription and translation of a DNA copy in which an 87-bp-long deletion in the coding sequence for the 24-kd polypeptide was introduced revealed that the 24-kd polypeptide bears the proteolytic activity involved in the primary cleavage of the B RNA-encoded polyprotein.
双股 cDNA 由豇豆花叶病毒 B 组份 RNA 合成，并克隆到适当的载体中。用 4 个代表 B RNA 全部序列的克隆，构建了全长 DNA 拷贝，随后把它定位在噬菌体 SP6 或 T7 启动子的下游。用 SP6 或 T7 RNA 聚合酶从这个全长 DNA 拷贝转录的 RNA 分子，在兔网织红细胞裂解物中被有效地翻译成与从病毒 B 组份分离的 RNA 相似的 200kD 多肽。而且这种多肽很快被切割成 32kD 和 170kD 多肽，与病毒 B RNA 编码的 200kD 多肽完全一样。在体外，对一个 DNA 拷贝进行转录和翻译，该 DNA 拷贝的编码 24kD 多肽的序列中有 87 个核苷酸的缺失，结果表明 24kD 多肽具有参与 B RNA 编码的多蛋白初次切割的蛋白水解活性。
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