烟草花叶病毒运动蛋白cDNA的克隆及融合蛋白的表达

Cloning of Movement Protein cDNA of Tobacco Mosaic Virus and Its Expression in Escherichia coli

从烟草花叶病毒 (TMV)中提取总RNA ,通过反转录 PCR (RT PCR)扩增得到其运动蛋白 (MP)的基因 ,将扩增产物克隆到pMD 18T载体上。DNA序列分析表明 ,所得到的运动蛋白的基因全长为 80 7bp (GenBank接受号AY30 0 16 1) ,与已发表TMV序列 (GenBank登陆号为NC- 0 0 136 7)和同属的番茄花叶病毒 (ToMV ,GenBank登陆号为NC- 0 0 2 6 92相比核苷酸的同源性分别为 98 0 %和 80 9% ,氨基酸的同源性分别为 99 1%和 80 0 %。将目的片段亚克隆到表达载体pET 30a上 ,并在大肠杆菌JM10 9中诱导表达 ,诱导 9h后 ,融合蛋白表达量最大。诱导后的工程菌超声后经SDS PAGE检测 ,融合蛋白以可溶形式存在。

The movement protein gene was obtained by RT-PCR from the total RNA of tobacco mosaic virus and cloned to pMD-18T vector. Its length was 807 bp, compared with TMV and ToMV in GenBank, the identities of neucleotides and amino acids were 98.5%, 80.9% and 99.1%,80.0% respectively. The target fragment was subcloned to pET-30a, an expression vector. The fusion protein was expressed in the stain JM109 of Escherichia coli. The highest amount of expression time of the fusion protein which was solvent after sonication, was 9h after inducing.