甜菜坏死黄脉病毒75kDa通读蛋白基因构建与表达

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

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BNYVV75kDa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54kD。片段分别克隆到pJW2上,构建了这两个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。

The RNAs was extracted from purified beet necrotic yellow vein virus (BNYVV) isolated from Inner Mongolia of China. The first strand of cDNA encoding 54kDa fragment of 75kDa readthrough protein was synthesized from viral RNA template using reverse transcription, and 1. 5kb fragment was obtained after 30 PCR amplification cycles. The restriction map of the target fragment cloned into pGEM-7Zf(+) and its whole sequence has been analyzed. The result shows 54kDa fragment of 75kDa readthrough protein gene of BNYVV isolated from Inner Mongolia of China has 1509nts. Comparaing with published F13 isolate,this fragment was deleted with 3nts and shares 94. 97% and 96- 42% homology in terms of nucleotide sequence and deduced amino acid sequence respectively.