遗传性铁粒幼细胞贫血致病的ALAS2基因表达载体的构建  

Construction of Recombinant Vector Expressing ALAS2 gene in X- linked Sideroblastic Anemia

作　　者：王逸群[1] 朱平[1] 石永进[1] 顾江英[1] 卜定方[1] 刘辉[1] 张英[1] 

机构地区：[1]北京大学第一医院血液科,北京100034

出　　处：《中国实验血液学杂志》2004年第5期687-693,共7页Journal of Experimental Hematology

摘　　要：X连锁遗传性铁粒幼细胞贫血 (XLSA)是红系特异性δ 氨基 γ酮戊酸合酶 (δ amionlevulinicacidsyntase 2 ,ALAS2 )基因突变造成的 ,本研究构建ALAS2基因的真核表达载体 ,观察表达载体转染真核细胞后蛋白表达的情况。将获自人胎肝的ALAS2基因插入到红色荧光蛋白质粒 pDs red2 N1中 ,命名为pDs red2 N1 ALAS2 ;采用电穿孔方法将质粒转染K5 6 2细胞 ;转染后 4 8小时提取细胞总RNA ,进行RT PCR检测ALAS2基因表达 ;流式细胞术检测红色荧光蛋白表达。再采用脂质体方法将质粒转染COS7细胞 ,转染后 72小时提取细胞总RNA ,RT PCR检测ALAS2基因表达 ,荧光显微镜观察COS7细胞红色荧光蛋白表达情况。结果表明 :构建了一种 pDs red2 N1 ALAS2真核表达载体 ,提取质粒DNA经酶切、电泳可以看到在 4 70 0bp和176 4bp处存在两条电泳条带 ;全长测序证实构建的载体序列正确 ;质粒转染K5 6 2和COS7细胞后均出现阳性ALAS2基因转录产物 ;流式细胞术检测转染后K5 6 2细胞红色荧光蛋白表达的阳性细胞百分率为 19.2 % ;荧光显微镜显示转染后COS7细胞红色荧光蛋白 ,表达高峰出现在转染后第 3天 ,阳性细胞百分率为 10 .7% ,并可持续表达 10天左右。红色荧光蛋白的表达可以间接说明ALAS2基因的表达。结论X-linked sideroblastic anemia (XLSA) is caused by mutations of erythr oid-specific 5-aminolevulinate synttase (ALAS2) gene. In this study a eukary oti c expression vector of ALAS2 was constructed and transfected into eukaryotic cel ls to observe th e expression of ALAS2 gene. The full length cDNA of ALAS2 gene was in serted into plasmid pDs-red2-N1, named pDs-red2-N1/ALAS2. Then, the vector w as transfected into K562 cells via electroporation. At 48 hours after transfection, total RNA from K562 cells was extracted, expressio ns of ALAS2 gene and protein with red fluoreseence in the K562 cells were detected by RT-PCR and flow cytometry, respectively. The vector was also transfected into COS 7 cells via liposome. Both mRNA and protein expre ssion in COS7 cells were detected by RT-PCR and fluorescence microscopy. The re sult showed that after the pDs-red2-N1/ALAS2 eukaryotic expression vecto r was digested by KpnI and BamHI, two fragments of 4 700 bp and 1 764 bp were displayed by el ectrophores is on agarose gel. Sequence method confirmed that the sequence was correct. RT- PCR amplified the total RNA ex tracted from the transfected K562 and COS7 cells, and could find mRNA of ALAS2 g ene that can′t be found in K562 and COS7 cells usually. The expressions of bot h fluorescein and ALAS2 were significantly increased. The percentage of positive cells reached ab out 19.2% and 10 7%, respectively. ALAS2 expression lasted for 10 days in COS7 cells and the peak was at the third day. It is concluded that the eukaryotic expr ession vector of ALAS2 gene is successfully constructed; K562 and COS7 cells tra nsfected with t he vector via electroporation and liposome can express ALAS2 protein. So, the v ector has the potential in gene replacement and can be used for patients with XLSA in future.

关 键 词：遗传性铁粒幼细胞贫血 ALAS2基因 载体构建 基因转染 基因治疗 

分 类 号：R556.9[医药卫生—血液循环系统疾病]