甜菜坏死黄脉病毒75kDa通读蛋白基因构建与表达

利用DNA重组技术，将甜菜坏死黄脉病毒(BNYVV)内蒙分离物的CP基因和54kDa通读区片段拼接。构建了BNYVV 75kDa通读蛋白基因。序列分析表明，构建的75kDa通读蛋白基因与野生型相比．只有4个核苷酸发生了改变(包括将CP基因的终止密码子TAG改造为ATG)，相应地2个氨基酸也发生了改变。将75kDa通读蛋白基因及其54kDa片段分别克隆到pJw2上，构建了这两十基因的原核表达载体。SDS—PAGE和western blotting检测结果表明，75kDa通读蛋白基因在E coli BL21(DE3)中经温度(42℃)诱导后除可特异地表达75kDa蛋白外。还产生两种小蛋白。75kDa通读蛋白基因的54kDa片段只表达出37kDa的蛋白。

Construction and Expression of 75kDa Readthrough Protein Gene from Beet Necrotic Yellow Vein Virus

Using DNA recombinant technique, coat protein (CP) gene and 54kDa fragment from beet necrotic yellow vein virus (BNYVV) RNA2 were ligated to construct 75kDa readthrough protein gene. Comparing with wild-typed 75kDa readthrough protein gene, 4 nucleotides of constructed 75kDa readthrough protein gene were replaced, including CP amber termination codon TAG to ATG. Corresponding, 2 amino acids were changed. The temperature-inducible expression vectors containing 75kDa readthrough protein gene or its 54kDa fragment were constructed and transformed into E. coli BL21 (DE3) respectively. The results of SDS-PAGE and Western blotting show: (1) specific expression of 75kDa readthrough protein gene was achiyed by temperature induction. Two smaller proteins, one is 37kDa. were also observted. (2) only a 37kDa protein from 54kDa fragment was expressed. This is the first report about specific expression of BNYVV 75kDa readthrough protein gene in E. coli.