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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 BNYVV75kDa avirulence gene was constructed by splicing the CP gene of the Inner Mongolia isolate of beet necrotic vein virus (BNYVV) with the 54kDa gene fragment using DNA recombination technology. Sequence analysis showed that only 4 nucleotides of the 75kDa gene were constructed, including the modification of the TAG of the CP gene to ATG. Correspondingly, 2 amino acids were also changed. The 75 kDa readonin gene and its 54 kD. The fragments were cloned into pJW2 respectively, and the prokaryotic expression vectors of these two genes were constructed. The results of SDS-PAGE and Western blotting showed that the 75kDa gene could express 75kDa specifically in E. coli BL21 (DE3) after induced by temperature (42 ℃). Protein, but also produce two small proteins. The 54 kDa fragment of the 75 kDa readonin gene only expresses a 37 kDa protein.