家蚕微孢子RAD51基因的克隆、原核表达与体外翻译

Cloning and Characterization of a Gene Coding for RAD51 From Nosema bombycis

从家蚕微孢子 (Nosemabombycis)中通过RT PCR扩增出RAD5 1(DNArepairprotein)基因的 3′端部分序列 ,经5′ RACE(RapidAmplificationofcDNAEnds)后得到全长cDNA序列 ,共 10 0 2bp ,编码 334个氨基酸。虽然用NorthernBlot检测不出mRNA的特异性表达带 ,但用RT -PCR直接扩增出全长cDNA而证实了该基因。该cDNA已作为新基因提交到GenBank数据库中 ,登录号为AF0 3730 5。将该基因在大肠杆菌 (E .coli)中进行表达 ,得到表达量较高的分子量约 37ku的蛋白质 ,经亲和层析纯化后得到较浓的单一蛋白带 ,纯化效果较好。用兔网织红细胞裂解液在体外也翻译出该分子量为 37ku的蛋白质 ,进一步确证了该蛋白。通过BLAST查询GenBank数据库 ,发现它与许多生物体的RAD5

The RAD51 gene is an eukaryotic counterpart of the Escherichia coli RecA gene,which is involved in genetic recombination and in repair of double strand DNA breaks.We have identified a RecA/RAD51 like gene (RAD51) from Nosema bombycis by RT PCR and 5′ RACE.The predicted 334 amino acid protein showed significant homology to RAD51 gene in Bombyx mori,M.musculus,X.laevis,S.cerevisiae and RecA gene in E.coli .In this paper we reported this gene in Nosema bombycis and detected it by RT PCR.We translated the gene products in vitro and got the expected size of protein.We also expressed and purified the protein of this gene.