荧光差异显示PCR克隆大鼠脑缺血相关基因KIAA0280

Identification of Rat Ischemia Related Gene KIAA0280 by Fluorescence Differential Display PCR

【目的】研究大鼠急性局灶性脑缺血时缺血组织与非缺血组织中基因表达的差异,克隆出与脑缺血相关的基因。【方法】应用荧光差异显示PCR(FDDPCR)从同一例大鼠大脑皮层缺血组织和非缺血组织中筛选出有表达差异的基因片段,经反向Northernblot杂交,将杂交阳性的片段且经RT-PCR反复验证,选取差异明显的片段进行克隆测序;经生物信息学分析处理,选取同源性低的EST片段R6,利用cDNA5'端快速扩增PCR(5'RACE法)进行基因全长的克隆。【结果】利用5'RACE法成功地克隆EST片段R6至编码区,扩增并获取全长4821bp及完整的可读框(ORF),RT-PCR及Northernblot印迹结果证实了克隆的结果,同源性分析发现该基因ORF与人大脑中已知的KIAA0280基因高度同源,同源性96%,编码166个氨基酸,为结构及功能未明确蛋白质,该基因定位于大鼠第11号染色体长臂(11q.13)。【结论】应用荧光差异显示PCR成功地自大鼠体内克隆到与人KIAA0280相似的基因,其在大鼠急性局灶性脑缺血中明显上调,该蛋白质结构及功能均未见报道及研究,其显著地差异表达提示其在脑缺血病理过程中具有重要作用。

To study the differences in gene between acute focal ischemia and non ischemia tissues in rat so as to clone the related gene of brain ischemia. The fluorescence differential display was applied to screen the differential expression gene between focal brain ischemia and non ischemia region. After identification by reverse Northern blot and RT PCR, the full length of the target EST R6 that was not highly homologous with the known gene was cloned by 5′RACE. R6 in 4 821 bp full length was successfully cloned by 5′RACE and the open reading frame (ORF) was obtained. The results of Northern blot and RT PCR were accorded with the cloned results. Homologous analysis showed that ORF of R6 was highly homologous (96%) with human brain gene KIAA0280, which encoded 166 amino acids. Nevertheless, the protein structure and function of KIAA0280 was unknown. This KIAA0280 gene was located in 11th Chromosome (11q.13) of rat. [Conclusion]The gene similarity to KIAA0280 is successfully cloned by fluorescence differential display from rat focal brain ischemia. It is up regulated in rat focal ischemia. The structure and function of KIAA0280 has not been reported. Its markedly differential expression suggests that it plays an important role in the pathogenesis of brain ischemia. $$$$