血红素合成酶特异性RNAi表达载体的构建及其干扰效果鉴定

目的利用含红色荧光报告基因（DsRed）的pGCsi-U6／Neo／DsRed／质粒构建干扰人红系特异的γ-氨基-6-酮戊酸合成酶（eALAS）基因表达的短发夹RNA（shRNA）表达载体，并进行活性鉴定。方法根据GenBank中eALAS的序列，设计3条表达shRNA的互补DNA序列，分别克隆至质粒载体pGCsi-U6／Neo／DsRed中构建重组质粒，转化大肠杆菌DHSa菌株扩增后，提取质粒测序分析，以相同的方法构建并鉴定eALAS基因的真核表达载体pEGFP-N1-eALAS；将3种pGCsi-U6／Neo／DsRed／eALAS-shRNA分别与pEGFP-N1-eALAS共转染HEK293细胞株，通过流式细胞仪检测pEGFP-N1-eALAS融合蛋白的荧光强度，选择最佳RNA干扰（RNAi）靶点，并在基因和蛋白水平检测eALAS的表达，进一步验证最佳RNAi靶点的敲减效率。结果经测序，模板序列与设计序列完全正确，最佳RNAi靶点敲减效率达72．45％，eALAS在基因和蛋白水平显著下降。结论成功构建了eALAs特异性RNAi表达载体，为下一步研究铁效应元件eALAS在铁代谢异常所致红系统疾病的机制奠定了基础。

Construction and interfering effect identification of RNAi eukaryotic expression vectors of erythrocyte Aminolevulinic acidsynthetase mRNA

Objective To construct the recombinant plasmid expressing three eALAS -targeted short hairpin RNA （shRNA） with pGCsi-U6/Neo/DsRed plasmids vector, detected the interfering effect in HEK293 cell after cotransfection with the eukaryotic vector of the eALAS gene. Methods Three pairs of cALAS DNA sequences were designed according to the eALAS sequence in GeneBank. The sequence was cloned into with pGCsi-U6/Neo/DsRed and then sequence analysis was performed, and used the same method to construct and identify eALAS eukaryotic expression vector pEGFP-N1- eALAS. Recombinant plasmids of three pGCsi-U6/Neo/DsRed/shRNA-eALAS and a pEGFP-NI-eALAS were cotransfected into HEK293 cell by Lipofectamine2000 respectively, the optimal vector of three shRNA was chosen by flow cytometry testing the fluorescence of pEGFP-NI-eALAS fusion protein, which RNAi inhibitory effect was further confirmed by RT-PCR and Western blot checking eALAS in the level of gene and protein. Results The specific shRNA and eALAS coding sequences were consistent with the designed fragments, the inhibtive effect of the optima shRNA vector on the expression of eALAS is up to 72.45% by flow cytometry, and the level of eALAS decreasd significantly by RT-PCR and Western blot. Conclusions The RNAi eukaryotic vector pGCsi-U6/Neo/DsRed/shRNA-eALAS of eALAS gene is constructed successfully,which can provide a valid tool to research the association between the abnormal iron metabolismred and blood cell disorders.