黑木耳核糖体失活蛋白cDNA 3′-端的克隆与分析

为了克隆黑木耳核糖体失活蛋白cDNA 3′-端,根据随机测得的中间一段蛋白质序列设计简并引物,应用RT-PCR和3′-RACE反应方法,将得到的基因片段与质粒连接,并转化至大肠杆菌中,进行蓝白筛选,再通过琼脂糖凝胶电泳法和PCR法验证白斑,从而得到阳性重组质粒,最后克隆出该目的蛋白基因片段。结果表明,克隆出的cDNA 3′-端为330bp的开放阅读框(ORF),编码107个氨基酸和2个终止密码子。经试验证明和文献检索,克隆得到的cDNA 3′-端为一种新基因。

The Cloning of 3'-end of cDNA of Ribosome-inactivating Protein from Auricularia auricular

To clone the 3′- end of cDNA of RIP from Auricularia auricular, this study firstly designs degenerate primer according to its middle amino acid sequence which had been random determined, then connects obtained gene - fragment and plasmid applying methods of RT - PCR and 3′-RACE reaction, makes transformation into E. coli and going on blue -white screening, finally, gains positive recombinant plasmid pass checking of white - spot by methods of agarose gel electrophoresis and PCR, thus, has successfully cloned the gene - fragment of interest protein. The results indicate that the 3′- end of cDNA is an open reading frame of 330bp which contains 107 amino acids and 2 nonsense codons. By testifying from experiments and literature retrieval, the obtained 3′-end of cDNA is a new gene.