大鼠肝再生中1个表达上调新基因的克隆表达及分析

The expression and analysis of the cloning of a up-regulated novel gene during liver regeneration in rat

目的 克隆肝再生相关新基因。方法 以抑制性消减杂交得到的1个EST为模板制备探针,运用Southern印迹方法,从构建的再生肝76 h c DNA文库中调取了它的c DNA全长,并利用基因原核表达技术,表达并纯化出它的蛋白产物,制作了该蛋白的兔源多抗,分别用所制该基因的探针,采用点杂交技术检测了0～76 h的再生肝材料。结果 获得了1个新基因全长,BL AST检索结果为1未知功能基因,暂时命名为liver regeneration relatedprotein(L RRP) ,大鼠基因组数据库中检索结果证实L RRP位于19q12且由4个外显子和3个内含子组成,递交Gen Bank获登录号为AY0 98917。表达纯化出了它的融合蛋白产物,并得到了它的多克隆抗体。点杂交结果也证实了基因在肝切除后76 h明显上调。结论 L RRP基因全长的克隆和它的高效多克隆抗体的获得为进一步研究该基因的功能奠定了基础。

Objective To clone and characterize a novel gene which might be involved in liver regeneration.Methods A 76 h regenerative liver tissue cDNA library was constructed by SMART technology.One EST from suppression subtractive hybridization was probed by Dig and carried out phage in situ hybridization to clone positive clone from 76h cDNA library;Construction of expressed vector was made and the specific expressed protein was purified,and its multiclone antibody was produced by injecting fusion protein products to rabbit.Dot blot technique monitored its mRNA variation during 0～76 h.Results A novel full-length gene whose function was unknown by BlAST examination was cloned successfully, and was called temporally liver regeneration related protein (LRRP) and deposited in GenBank (accession NO.AY098917). The results that blasted in RGD (rat genome database) showed that it was localized in 19 q12 and was composed of four extrons and three introns.Dot blot results indicated that its expression quantity increased obviously in 76 h after hepatectomy.Conclusion The cloning of LRRP gene and its multiclone antibody provides a good base for the studies of liver generation related protein.