甜菜夜蛾核多角体病毒泛素基因的克隆及原核表达

Cloning and Prokaryotic Expression of Ubiquitin Gene of Spodoptera exigua multi-nucleocapsid nucleopolyhedrovirus

甜菜夜蛾核多角体病毒(Spotoptera exigua multi-nucleopolyhedrovirus,SeMNPV)泛素基因ubiquitin被克隆和序列分析.该基因编码区全长243bp,编码80个氨基酸残基,预计蛋白质分子量为9.4kDa。将这一ubiquitin基凶克隆到原核表达载体pET-28a上.转化至BL21 (DE3)中,用IPTG进行诱导表达,对表达的条件进行了优化。用异源的泛素单克隆抗体检测目的蛋白,Western blot实验证明所表达的蛋白是泛素蛋白。同时,我们制备了特异性的抗体,为以后的研究工作做了基础。通过计算机软件Gendoc对不同来源的泛素进行分析,结果显示,病毒中的泛素与真核细胞中的泛素相比较,泛素的氨基酸序列有较大的变化,杆状病毒的泛素基因在分子进化上可能有比较独特的途径。

A ubiquitin gene of Spotoptera exigue multi-nucleopolyhedrovirus (SeMNPV) was cloned and sequenced. The result showed that the gene size was 243 bp, encoded 80 amino acids with apredicted size of 9.4kDa. To construct expressive vector for the udiquitin gene of SeMNPV, the fragment containing ubiquitin gene was inserted into pET-28a expressive vector. Expression of the fusion protein was induced by IPTG in E.coli BL21 (DE3). By changing the time induced and the content of IPTG, the fusion protein had an optional expression level. The fusion protein was identified by Western blot with a mouse Ab anti Ubiquitin from bovine oringe. To do the further study, the specific Ab anti viral Ubiquitin was produced. In addition, the sequence of amino acids was analyzed with the software of Gendoc. The result displayed that ubiquitin of baculoviruses had some different amino acid substitutes in some residues and the amino acid sizes varied a lot compared with eukaryotic udiquitin, From these, we reckon that ubiqtuitins of baculovirus may have a unique way in the gene evolution.