日本囊对虾Ran基因的克隆表达与蛋白质GTP结合活性分析

在对虾抑制性差减杂交（suppression subtractive hybridization,SSH）研究过程中,首次发现一段经同源比较为Ran基因的部分序列,在抗病对虾中上调表达.为了进一步探索对虾Ran基因的功能,通过RACE-PCR的方法克隆得到了对虾Ran基因全长共1441个碱基,其中开放阅读框为645个碱基,共编码215个氨基酸,这是首次在海洋无脊椎动物体内克隆到该基因.本研究还将该基因克隆到原核表达载体PGEX-4T-2中并转化大肠杆菌BL21,37℃下诱导6h,超声裂解表达菌株,结果表明GST-Ran融合蛋白在大肠杆菌中为可溶性表达,蛋白大小约为50ku,经纯化得到了纯度大于90%的GST-Ran融合蛋白.随后的GTP结合试验验证了Ran蛋白具有GTP结合活性.

Cloning, Recombinant Expression and GTP-binding Activity Analysis of Ran Gene of Penaeus japonicus

In previous studies,a cDNA fragment was found to be highly homologous with ras-related nuclear proteins （Ran proteins） by suppression subtractive hybridization （SSH） using WSSV-resistant shrimp. In this investigation,Ran gene was cloned from Marsupenaeus japonicus shrimp by RACE-PCR,the total cDNA was 1 441 base pair and the ORF of shrimp Ran gene contained 645 bp,which encoded 215 amino acids. This was the first Ran gene obtained from marine invertebrates. Then the gene was cloned in pGEX4T-2 and expressed in Escherichia coli BL21. After induction with IPTG at 37 ℃ for 6 h,the GST-Ran fusion protein （about 50 ku） was expressed and purified by glutathione-agarose beads. Based on protein domain analysis,the Ran protein was predicted to have a GTP/ATP binding motif. To test whether the Ran protein was endowed with GTPase activity,the GTP-binding assay was performed. The result revealed that the Ran protein had the GTP-binding activity.