人Dp71 shRNA载体的构建与鉴定

[Construction and evaluation of human Dp71 shRNA vector].

作者信息

Tan Sichuang, Chen Zhikang, Cao Xiaoxia, Wen Qiaocheng, Zhang Weilin, Zeng Qingren, Tan Sipin

机构信息

Department of Cardiovascular and Thoracic Surgery, Second Xiangya Hospital, Central South University, Changsha 410011,China.

出版信息

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2014 Apr;39(4):338-43. doi: 10.3969/j.issn.1672-7347.2014.04.003.

DOI:10.3969/j.issn.1672-7347.2014.04.003

PMID:24820271

Abstract

OBJECTIVE

To construct effective short hairpin RNA (shRNA) recombinant plasmids targeting human Dystrophin Dp71 gene, and evaluate their interference efficiency.

METHODS

Three pairs of siRNA sequences targeting human Dp71 gene and one pair of control siRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. The shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Dp71-shRNA and control shRNA plasmids were transfected into human normal gastric epithelial cells (GES-1) and human bronchial epithelium (HBE). Western blot was used to evaluate its interfering efficiency.

RESULTS

Restriction enzyme digestion and sequencing showed that the Dp71-shRNA vectors were successfully constructed. Western blot displayed that Dp71 protein expression was reduced to a significant degree after transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most efficiently.

CONCLUSION

Dp71-shRNA vectors have been successfully constructed. The 3 Dp71-shRNA plasmids can inhibit Dp71 expression in GES-1 and HBEC, with Dp71-shRNA2 plasmid displaying the highest inhibition efficiency.

摘要

目的

构建靶向人抗肌萎缩蛋白Dp71基因的有效短发夹RNA（shRNA）重组质粒，并评估其干扰效率。

方法

设计、合成三对靶向人Dp71基因的小干扰RNA（siRNA）序列和一对对照siRNA序列，然后将其插入pRNAT-U6.1/Neo载体。通过酶切和测序对shRNA重组载体进行评估。将Dp71-shRNA和对照shRNA质粒转染到人正常胃上皮细胞（GES-1）和人支气管上皮细胞（HBE）中。采用蛋白质免疫印迹法评估其干扰效率。